Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 3, Num. 1, 1992

BIOLOGICAL AND CHEMICAL CHARACTERIZATION OF THE ANTIBIOTIC ACTIVITY OF STREPTOMYCES SPECIES ISOLATED FROM GRAPEVINE CARPOSPHERE

A.VERCESI, G.NASINI(1)and R.LOCCI(2)

Institute of Plant Pathology, University of Milan, (1)Department of Chemistry, CNR Centre for Organic Natural Substances, Polytechnic of Milan and (2)Chair of Mycology, University of Udine, Italy

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The ecology of Streptomyces spp. has been studied mostly in reference to soil, where the genus is ubiquitous. Here their activities include degradation of animal, plant and microbial residues and possibly biological control (Williams et al., 1984).

On the other hand very little is known about their role in other environments. The present report deals with an analysis of the antibiotic activity of streptomycetes, isolated from the grapevine carposphere (Vercesi et al., 1990, 1991), against other organisms from the same habitat and the characterization of the active metabolites.

MATERIALS and METHODS

Antibiosis. The antibiotic activity of the 220 actinomycetes isolated from grapevine (Vitis vinifera, cv. Riesling) carposphere (Table 1) was tested against 107 yeasts, associated with grapevine sour rot and grouped according to numerical methods (Vercesi et al., 1986) and against nine mycelial fungi, all isolated from grapes (Tables 2 and 3).

The streptomycetes were grown on CYA (Czapek agar + 0.2% yeast extract), except for S.cyaneus, S.phaeochromogenes and Actinoplanes sp., which were cultivated on PDA (potato dextrose agar), in 9 cm diameter Petri dishes (25 ml of medium) for 14 dd at 26 C. Yeasts were grown for 3 dd on PDYA (PDA + 0.5% yeast extract) and mycelial fungi for 10 dd on CA (Czapek agar) at 24 C. A distilled water suspension of yeast cells and fungal conidia was used to inoculate 9 cm diameter Petri dishes containing 25 ml of PDYA and CA. Agar mycelium disks (0.5 cm in diameter) of actinomycetes were placed on the surfaces of the agars inoculated with the test yeast and mycelial fungi. Inhibition zones were determined after 3 and 7 dd for yeasts and after 7 and 14 dd for fungi.

Characterization of the metabolites. In the course of a preliminary investigation S. albidoflavus strains were grown on PDA and CYA. Subsequently strain R81 was grown on Czapek broth + yeast extract (0.2%) and on Beecham's medium (Williams et al., 1983). The strains of S.cyaneus and S.phaeochromogenes and Actinoplanes sp. were grown on PDA in Roux flasks. All strains were incubated at 27 C for 14 dd. Secondary metabolites were extracted with EtOAc:MeOH (100:1), which was poured over the solid medium and then left overnight on solid media or, in the case of liquid media, stirred for 2-3 h.

-------------------------------------------------------------
Taxon          No. of                   Strains 
               Strains 
-------------------------------------------------------------
Streptomyces       117    ACT 1-4, 10, 11, 13, 18, 24, 26,
albidoflavus              37,39, 40, 43, 44, 46, 52, 53, 56 
                          R 2-7, 9-11, 14-17, 19-29, 31-33,    
                          35-39, 41-48, 52, 53, 55, 56, 59-61,

                          63-65, 67, 68, 70, 73, 76-78,80, 81, 
                          83, 85-90, 92, 93, 97-100, 102, 105, 
                          109-111, 113, 114, 120, 126, 128,    
                          131-134, 142-144, 148, 149, 152,     
                          153, 155, 156, 1,58, 162 
S.albus             1     ACT 22 
S.atroolivaceus     3     R 1, 51, 129 
S. chromofuscus     12    ACT 6-8, 27 
                          R 8, 12, 30, 54, 58, 82, 91, 112 
S.cyaneus           5     R62, 121, 147, 1,54, 161 
S.diastaticus       10    ACT 47, 51, 57 
                          R 18, 69, 72, 74, 106, 139, 145 
S.exfoliatus        8     R 13, 125, 127, 135, 136, 138, 140,  
                          141 
S.griseoruber       12    ACT 15, 16, 25, 28, 38, 49, 50 
                          R 34, 101, 107, 117, 150 
S.phaeochromogenes  2     R 95, 137 
S.rochei            9     ACT 20,23 
                          R 50, 71, 75, 84, 103, 122, 146 
Streptomyces spp.   40    ACT5,9, 12, 14, 17, 19, 21, 29-36,   
                          41, 42,45,48,54,55,58 
                          R 49, 57, 66, 79, 94, 96, 104, 108,  
                          115, 116, 118, 119, 123, 124, 157, 
                          159, 160 
Actinoplanes  sp.   1     R 130
--------------------------------------------------------------

Extracts were evaporated under vacuum and the residue dissolved in CH2Cl2. Silica-gel thin layer chromatography (TLC) of the extracts was developed with hexane:EtOAc (2:1) or with CH2Cl2: MeOH (9:1).

Spots were revealed with an oxidative solution containing Ce(SO4)2, 10g; H3[P(MO3O10)4], 25g; H2SO4, 60ml; water 940ml. Unnsprayed thin layer (TL) chromatograms were used to test antibiotic activity of the extracts by means of bio- autoradiography. Yeast strain L8O was suspended in potato dextrose broth + yeast extract and sprayed on TL chromatograms. Following 24 h incubation at 26 C, inhibition zones were detected by using a 0.1% solution of trypan blue in lactophenol.

Extract components were separated by column chromatography and characterized by UV (Jasco-Uvidec-510), IR (Perkin-Elmer 177), MS (Finnigan-MAT TSQ70) and 1H-NMR (Bruker CPX-300). Samples of antimycin A, b-rubromycin and isoflavones were supplied by Boehringer, Lepetit and Inverni della Beffa respectively.

------------------------------------------------------------
Cluster   No. of    Strains 
          Strains 
------------------------------------------------------------
A          21        L1,2,20-23,28,41,44-46,63 
                     74, 79, 82, 85, 87, 88, 96, 99, 101 
B          67        L 3-13, 15-19, 24-27, 29-35, 37 40, 42,   
                     43, 50, 52, 53, ,58-62, 68-72, 77, 80,    
                     81, 83, 84, 8,6, 89-93, 97,100, 102-107,  
                     109, 110 
C          19        L 36, 47-49, 51, 54-57, 64-66, 73, 75,    
                     76, 78, 94, 95, 108
-------------------------------------------------------------

---------------------------------------------------------
Taxon                   No. of              Strains 
                        Strains 
---------------------------------------------------------
Acremonium sp.             2            P271, P288 
Aspergillus sp.            1            P1235 
Aureobasidium pullulans    1            P1192 
Botrytis cinerea           1            P1238 
Cladosporium sp.           2            P1234, P1243 
Penicillium sp.            2            P1265, P1305
---------------------------------------------------------

------------------------------------------------------------Activity                         Actinomycetes 
against    -------------------------------------------------
                        Total        S. albidoflavus
cluster
                      No.    %              No.    % 
-------------------------------------------------------------
Some yeasts          102    46.4           68     58.1 
>/= 30% of yeasts    2B     12.7           23     19.7 

Some fungi           104    47.3           67     57.3 
>/= 30% of fungi     64     29.1           48     41.0
-------------------------------------------------------------

RESULTS

1. Antagonism.

One-hundred and forty actinomycetes, i.e. 63.6% of the tested strains, show some degree of inhibitory activity against either the yeasts or the fungi isolated from the grapevine carposphere.

One-hundred and two strains (Table 4) are active against the yeasts (68 S. albidoflavus, 14 Streptomyces sp., 5 S. cyaneus, 5 S. exfoliatus, 2 S.chromofuscus, 2 S.phaeochromogenes, 2 S.rochei, 1 S. albus, 1 S.atroolivaceus, 1 S. diastaticus, 1 Actinoplanes sp.).

Twenty-eight of the actinomycetes (23 S. albidoflavus, 2 S. cyaneus, 1 S.phaeochromogenes, 1 S. rochei, 1 Streptomyces sp.) inhibit at least 30% of all the yeasts tested.

Of these S.albidoflavus and S.rochei exhibit the highest activity against yeasts grouped in cluster C (Vercesi et al., 1986), S.cyaneus is more active against organisms in cluster B and S. phaeochromogenes against those of cluster A.

Antibiotic activity against mycelial fungi (Table 4) is shown by 104 actinomycetes (67 S.albidoflavus, 13 Streptomyces sp., 6 S.chromofuscus, 5 S.cyaneus, 4 S.rochei, 2 S.exfoliatus, 2 S. griseoruber, 2 S.phaeochromogenes, 1 S. atroolivaceus, 1 S.diastaticus, 1 Actinoplanes sp.), while 64 (i.e., 29.1%) of them (48 S.albidoflavus, 6 Streptomyces sp., 4 S.chromofuscus, 2 S.cyaneus, 2 S.phaeochromogenes, 1 S.rochei, 1 Actinoplanes sp.) inhibit 30% or more of the tested mycetes.

The genera Aspergillus, Cladosporium and Penicillium are most strongly inhibited. S.chromofuscus and S.phaeochromogenes do not affect Aureobasidium pullulans, the latter actinomycete does not inhibit Botrytis cinerea.

S.albidoflavus strains are active against most fungi, however activity against Acremonium is weak.

Among active strains, 29% inhibit both the yeasts and the mycelial fungi, 17.7% only the yeasts and 17.7% just the fungi.

Strains grouped in the S.albidoflavus cluster show the highest antibiotic activity. Sixty-eight of the 102 (66.7%) strains active against yeasts - and 23 out of 28 (82.1%) against at least 30% of the organisms - belong to the cluster.

Similarly 67 of the 104 (64.4%) strains active against mycelial fungi are members of the S.albidoflavus cluster. Representatives capable of inhibiting 30% or more of the fungi are 48 out of 64 (75%).

Eighteen S.albidoflavus strains inhibit more than half of the test organisms and six of them all the strains.

2. Metabolite characterization. Because of their high activity, S.albidoflavus, S.cyaneus, S.phaeochromogenes and Actinoplanes sp. were subjected to further investigations.

Figure 1. Mass spectrum of the antimycin complex.

All strains examined produced large amounts of lipids.

a) Streptomyces albidoflavus. Purification of the extract (PDA in 20 Roux flasks) gave a few milligrams of a compound with an [a]D value of +59.4 (CHCl3, c 0.1). The IR spectrum (liquid film) showed signals at 1745 (CO lactone), 1690 (CO) and 1650 cm^-1, the last band can be attributed to an amide function. The 1H-NMR spectrum showed the presence of one proton as a singlet to 8.55 5, attributable to an alde- hyde group, an ABX spin system due to three vicinal aromatic protons and three multiplets due to 3 CH-O systems; all the other protons are aliphatic. The sample when submitted to CIMS (Chemical Ionization Mass Spectrometry) (isobutane) showed molecular peaks (MH)+ at m/z: 562, 548, 534 and 520 (Fig. 1). All these data suggest that the sample is a complex of antimycins A, A1, A2 and A3 (Birdy et al., 1980; Kurtbtike et al., 1980). An accurate TLC and 1H-NMR comparison confirmed these findings.

Figure 2. Daidzein (R= H) and Genistein (R= OH).

b) Streptomyces cyaneus. Extracts (1.1g) of S.cyaneus, grown on PDA (40 Roux flasks) were treated with hexane (50 ml); the hexane layer was separated and concentrated to give 10 g of a mixture of triglycerides, while the residue (700 mg) was chromatographed with CH2Cl2-MeOH (15:1) as eluant to yield 200 mg of a crystalline yellow compound that showed UV, gamma max, nm: 246, 266, 334, 381, 400 and 424 and IR spectrum with a band at 1730 cm^-1 (ester CO); the ElMS (Electron Impact Mass Spectrometry) spectrum gave a molecular peak at M/z 334, not in agreement with the 1H and 13C NMR data. In fact FABMS (Fast Atom Bombardment Mass Spectrometry), m/z 641 (MH+) was consistent with a diglucoside substance whose aglycon moiety has a mass of 334. The physico-chemical data are in agreement with the structure of chartreusin (Fig. 3), an antibiotic produced by S.char- treusis (Leach et al., 1953) and which is the subject of renewed interest because of its antitumor activity (McGovren et al., 1977; Kon et al., 1990).

c) Streptomyces phaeochromogenes. This species, together with Actinoplanes sp., gave good yields of a red compound identified by 1H-NMR, FABMS and TLC comparison with rubromycin (Fig. 4) (Berdy et al., 1980), a condensed naphtoquinone antibiotic first isolated by Brockmann and Zeeck

Figure 3. Chartreusin.

Figure 4. b-Rubromycin.

The antagonistic activity of actinomycetes isolated from grapevine berries against yeasts and mycelial fungi inhabiting the same environment is surprisingly widespread.

More than half of the actinomycete population shows activity against either the yeasts or the fungi and more than a third inhibits them both. Six of the strains inhibit all tested organisms.

Strains grouped in the S.albidoflavus cluster are the most active antagonists.

The antibiotics isolated are not new. An item worth further investigation, is the large amount of fatty material (some showing inhibition haloes in the antibiotic plates) produced by some of the organisms.

As in previous studies (Chimura et al., 1975; Umezawa et al., 1975) isoflavones were isolated from streptomycetes grown on media containing plant organic nitrogen sources. However according to the present investigation, at least daidzein and genistein do not appear to be synthesized de novo by streptomycetes, since they were also isolated from soybean peptone, one of the medium ingredients, prior to inoculation. This possibility however was conceded also by the Japanese workers.

ACKNOWLEDGMENTS. The technical collaboration of Drs.A.Vegetti and E.Volpi and Ms T.Sprocati is gratefully acknowledged.

REFERENCES

Berdy. J., A.Aszalos, M.Bostian & K.L.McNitt (1980). CRC-Handbook of Antibiotic compounds. CRC Press, Inc., Boca Raton, Fl., Vol.2, pp. 378-387

Brockmann, H. & A.Zeeck (1970). Die Konstitution von a-Rubromycin, b-Rubromycin, g-Rubromycin and iso-g-Rubromycin. Chem. Ber., 103:1709-1726

Chimura, H., T.Sawa, Y.Kumada, H.Naganawa, M.Matsuzaki, T.Takita, M.Hamada, T. Takeuchi & H.Umezawa (1975). New isoflavones, inhibiting catechol-O-methyltransferase produced by Streptomyces. J.Antibiot., 28: 619-626

Kon, K., H.Sugi, K.Tamai, Y.Ueda & N. Yamada (1990). Synthesis and cytostatic activity of the antitumor antibiotic chartreusin derivatives. J.Antibiot., 43:372-382

Kurtboke, I., IR Cardillo, G.Nasini, B.Petrolini. P.Sardi & R.Locci (1985). Anti-Candida metabolites produced by streptomycetes isolated from the atmosphere of agricultural environments. The Actinomycetes, 19:223-231

Leach, B.E., K.M.Calhoun, L.E Johnson, C.M. Teeters & W.G.Jackson (1953). Chartreusin, a new antibiotic produced by Streptomyces chartreusis, a new species. J.Am.Che.Soc.,75: 4011-4012

McGovren, J.P., G.L.Neil, S.L.Crampton, M.I. Robinson & J.D.Douros (1977). Antitumor activity and preliminary drug disposition studies on chartrcusin (NSC 5159). Cancer Res., 37: 1666-1672

Umezawa, H., H.Tobe, N.Shibamoto, F.Nakamura, K.Nakamura, M.Matsuzaki & T. Takeuchi (1975). Isolation of isoflavones inhibiting DOPA decarboxylase from fungi and Streptomyces. J. Antibiot., 28:947-952

Vercesi, A., E.Volpi & R.Locci (1990). Preliminary investigations on the Streptomyces flora of grapevine berries. Actinomycetes, 1: 7-9

Vercesi, A., E.Volpi & R.Locci (1991). On the presence of Streptomyces spp. in the grapevine carposphere. Actinomyceles, 3:7-11

Vercesi, A., F. Zerbetto, M.Bisiach & R.Locci (1986). On the grouping of yeasts associated with grapevine sour rot by numerical methods. Ann. Microbiol., 36:23-34

Williams, S.T. M.Goodfellow, G Alderson, E.M.H.Wellington, P.H.A.Sneath & M.J. Sackin (1983). Numerical classification of Streptomyces and other related genera. J. gen. Microbiol., 129:1743-1813

Williams, S.T., S.Lanning & E.M.H. Wellington (1984). Ecology of actinomycetes. In: M.Goodfellow, M.Mordarski & S.T. Williams (eds.) The Biology of Actinomycetes. Academic Press, London, pp. 481-528.

Copyright 1992 CETA

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