Mycol. Res. 99 (8): 1007-1015 (1995) Prinled in Greal Brilain

Biologically active secondary metabolites of endophyticPezicula species

1007

B. SCHULZt"., J. SUCKER\ H. J. AUST\ K. KROHN2

, K. LUDEWIG2, P. G. JONES3 AND D. DORING 2

1 Inslitul fiir Mikrobiologie, Spielmannslr. 7, Technische Universiliil Braunschweig, D-38106 Braunschweig

2 Fachbereich Chemie und Chemiefechnik der Univ. GH Paderbom, Warburger Sfr. 100, D-33098 Paderbom

3 Insfifut fiir Organische Chemie, Technische Universifiit Braunschweig, D-38106 Braunschweig

Endophytic Pezicula strains were isolated from living branches of ten deciduous and coniferous trees and tested for their fungicidal,algicidal and antibacterial activities. From the three biologically active strains that were selected for fermentation, five substanceswere isolated and their structures determined as (R)-mellein, (- )-mycorrhizin A, 2-methoxy-4-hydroxy-6-methoxymethyl­benzaldehyde, (+ )-cryptosporiopsin and 4-epi-ethiosolide. They are strongly fungicidal and herbicidal, to a lesser extent algicidaland antibacterial. The synthesis of these metabolites in 85 isolates was dependent on conditions of culture and was also found to betaxonomically significant. The potential role of these substances is discussed with respect to mutualism and parasitism.

Some species of the Ascomycete Pezicula C Tu!. & TuLand its anamorph genus Cryptosporiopsis Bubak & Kabat areknown as endophytes (Petrini & Dreyfuss, 1981; Carroll &

Petrini, 1983; Sieber et al., 1989) and weak parasites of trees,including Pezicula livida (Berk. & Broome) Rehm, Peziculacarpinea (Pers.) Tu!. and Pezicula cinnamomea (DC) Sacco(Kehr, 1992). For P. livida the hosts are primarily conifers:Pinus sylves/ris L. (Scots pine), Picea abies (L.) Karsten (Norwayspruce), Abies alba Mill. (white fir) and Larix decidua MilL(European larch), but also the deciduous trees, Carpinus betulusL. (hornbeam), Fraxinus excelsior L. (white ash) and Fagussylvatica L. (European beech) (Kowalski & Kehr, 1992). As anendophyte, P. livida was, for example, isolated from 71 % ofthe peridermal bark samples of Pinus sylvestris and 50% of theperidermal bark samples of Picea abies (Kowalski & Kehr,1992). As a parasite it causes cankers on the trunks of its hosts(Kowalski, 1982; Butin, 1989). P. cinnamomea is particularlyknown for causing Pezicula canker of Quercus rubra L. (red oak)(Kehr, 1992), but was also isolated as an endophyte from all12 of the deciduous and coniferous trees that Kowalski & Kehr(1992) studied. The capability of a fungal strain to beendophytic for a period of time and later to become parasiticis not contradictory (Carroll, 1988; Sieber, Sieber-Canavesi &Dorworth, 1991; Kehr & Wulf, 1993). Potentially pathogenicstrains have frequently been isolated as endophytes (e.g.Cabral, Stone & Carroll, 1993; Schulz et al. 1993). Both P.livida and P. cinnamomea may remain asymptomatic for manyyears only becoming parasitic when their hosts are stressed(Butin 1989; Kehr, 1991, 1992; Kehr & Wulf, 1993).

• Corresponding author.

The question arises: to what extent is Pezicula mutualisticwith its host during the endophytic phase? One way in whicha fungus can assume a mutualistic role is to excrete metaboliteswhich are toxic for pathogens of its host (McCutcheon,Carroll & Schwab, 1993). That this is the case, at least in vitro,could be shown by Noble et al. (1991) for one species each ofPezicula and Cryp/osporiopsis. They isolated an echinocandinwith antimicrobial properties from extracts of cultures of thesefungi. In another in vitro experiment, Ohsawa & Katsuya(1987) showed that a Pezicula sp. that had been isolated fromlarch wood infected with butt-rot basidiomycetes was able toinhibit the decay of that wood. Previously, Fisher, Anson &

Petrini (1984) had found that an endophytic strain ofCryptosporiopsis isolated from Vaccinium myrtillus L. produced,in vitro, at least three antibiotic-containing fractions.

To test the hypothesis that Pezicula can produce antibioticsubstances as its contribution to a possible mutualisticrelationship, we first did confrontation tests to check forpotential antagonisms of Pezicula livida against Fusicoccumgalericulatum Corda, a colonizer of dying branches (Butin,1989) and a natural branch pruning fungus (R. D. Kehr,personal communication). We then tested extracts of thefungal cultures for antibiotic activity. Subsequently, thebiologically active substances were isolated and their structuresdetermined.

MATERIALS AND METHODS

Fungal isolates

All strains of Pezicula were isolated as endophytes fromasymptomatic branches of trees in Lower Saxony followingsurface sterilization (Kowalski & Kehr, 1992). The species and

Secondary metabolites of endophytic Pezicula species

Table 1. Pezicula strains and their hosts

1008

Species Strain number Host

P. acericola (Peck) SaccoP. alnicola j. W. GrovesP. carpinea

P. cinnamomea

P. livida

Pezicula sp.

191623411919,23482326,2327,23401917,1920,2331,2332,2365192819181927, 2366, 23681921,23402350,23462339,23421924,2333235523371925,23531922,2375,2379,23861926,2357235611561929,1931,2377,2381,23841930,1932,2370-2372,2374,2376,2378,23802382,2383,2385,2387,2388545,548,5542328,23292344,2347,23492338,23432334-23362358,2360,2363,23672351,2352,2354192323732359,2361,2362,2364553,2369

Acer pseudoplalanus L. (sycamore)Alnus glulinosa (L.) Gaertn. (black alder)A. pseudoplalanusCarpinus betulusFagus sylvalicaFraxinus excelsiorQuercus robur L.

Abies albaA. pseudoplalanusA. glulinosaBelula pendula Roth (silver birch)C. belulusF. sylvalicaF. excelsiorPicea abiesQ. robur L. (pedunculate oak)C. belulusF. sylvalicaP. abiesPinus sylvestris

unknownA. albaA. pseudoplalanusA. glulinosaB. pendulaC. belulusF. excelsiorP. abiesP. sylveslrisQ. roburunknown

The strains printed in bold type were used for subsequent fermentations.

hosts of the Pezicula strains used, as morphologically identifiedby Kowalski & Kehr (1992), are shown in Table 1. Strains 553,1156 and 2386 were fermented for isolation of the biologicallyactive substances. Fusicoccum galericulatum was isolated fromthe European beech, Fagus sylvatica. The fungal isolates werekindly provided by Dr R. D. Kehr, Professor H. Butin (Institutfur Pflanzenschutz im Forst, Biologische Bundesantalt fur Land­und Forstwissenschaft, Braunschweig, Germany) and DrT. Kowalski (Department of Forest Pathology, Faculty ofForestry, 31-425 Cracow, Poland).

Confrontation tests

P. livida (strains 545 - host unknown and 1156 - host F.sylvatica) and Fusicoccum galericulatum (also isolated from F.sylvatica) were inoculated simultaneously at opposite sides offour Petri dishes (MPY-agar medium) for each fungalcombination. After 39 d, growth of the isolates was evaluatedfor evidence of inhibition.

553 was cultured for 12-14 d at 20°C and strain 1156 for10-14 d at 17° in shaken culture (2000 ml Erlenmeyer flasks),each containing 800 ml of a 5 % (wIv) biomalt medium(Vitaborn, Hameln, Germany) at 125 rpm on a rotary shaker.Alternatively, strain 1156 was cultivated in a Braun fermenterfor 14 d in 5 % biomalt medium with the following setparameters: pH 6'3, O 2 = 90% saturation, 400 rpm and 25°.Strain 2386 was grown for 70 d at room temperature onMPY-semisolid medium (20 g 1-1 malt extract; 2'5 g 1-1yeast extract; 2'5 g 1-1 peptone from meat; 2'5 g 1-1 agar). Forcomparative thin-layer chromatography as autobiograms,strains 1917-1932 were grown on malt agar medium(10 g 1-1 malt extract; 12 g 1-1 agar) for 36 d at roomtemperature. For comparative thin layer chromatogramswithout autobiograms, all 85 Pezicula strains were grown onMPY-agar (as above but with 12 g 1-1 agar) for 28 d at roomtemperature.

Conditions of culture

Conditions of culture for fermentation were chosen to enablean optimal production of active secondary metabolites. Strain

Biological adivity, test organisms

The test organisms for the agar diffusion and screening testswere the bacteria Bacillus megaterium de Bary (gram positive)and Escherichia coli (Migula) Castellani & Chambers (gramnegative), the fungi Ustilago violacea (Pers.) Roussel (Usto-

B. Schulz and others

mycetes), Mycotypha microspom Fenner (Zygomycetes),Eurotium repens Corda (Ascomycetes) and Fusarium oxysporumSchltdl. (Deuteromycetes) and the alga Chiarella fusca ShihKrauss (Chlorophyceae), whereby inhibiton of C. fusca isusually correlated with broader herbicidal activity.

Screening for biological activity of fungal cultures

The Pezicula isolates were inoculated onto the appropriate solidmedia (in Petri dishes) for the respective test organisms. NB­medium (7'8 g 1-1 peptone from meat, Merck; 7'8 g 1-1

peptone from casein, Merck; 2'8 g 1-1 yeast extract, Oxoid;5'6 g 1-1 NaCl, D-( + )-glucose monohydrate, Merck; 12 g 1-1

agar, IMA) was used for the bacteria, MPY-medium (as above,but with 12 g 1-1 agar) for the fungi and CP-mediurn(10 g 1-1 yeast extract; 10 g 1-1 D-( + )-glucose mono­hydrate; 12 g 1-1 agar) for the alga. After attaining approx.3'5 cm diam., the colonies were sprayed with water sus­pensions of the test organisms. The cultures sprayed withbacteria were incubated for I d at 37°, those with fungi for3-5 d at room temperature and those with algae for 5-6 d at20° with 12 h of light: 12 h of darkness. The radius of the zoneof inhibition was measured.

Agar diffusion tests for biological activity of puresubstances

The substances to be tested were dissolved in the mosteffective solvent (mellein and epi-ethiosolide in methanol,cryptosporiopsin and mycorrhizin in acetone). Each solution(50 I-lI) was pipetted onto a sterile antibiotic filter disc(Schleicher & Schiill) and placed onto the appropriate growthmedium for the respective test organism and then sprayedwith that test organism as described above.

Seedling test for herbicidal activity of pure substances

Seeds of Lepidium sativum and Medicago sativa were placedonto filter paper in Petri dishes (4'5 cm diarn.) to each of which2'25 ml of sterile water and 0'25 ml of the dissolved substanceto be tested were added. The length of the seedlings wasmeasured and compared to the controls after 2 and 5 d andevaluated as % inhibiton of growth.

Instrumentation

Melting points were determined with a Biichi apparatus (DrTottoli) and are not corrected. Optical rotations were measuredwith a Perkin-Elmer 241 polarimeter. Infrared (lR) spectrawere obtained with a Nicolet 320 FT-IR spectrometer. Nuclearmagnetic resonance eH and 13C NMR) spectra were recordedwith a Bruker AM 400 (400 and 100 MHz, respectively).Chemical shifts were recorded in ppm (0) relative totetramethylsilane as internal standard (in CDCl3). Ultravioletjvisible spectra were recorded on a Beckman UV 5230 spectralphotometer. Mass spectra were obtained with a FinniganMAT 8430 mass spectrometer (70 eV). (p), (s), (t) and (g)

1009

designate primary, secondary, tertiary and guarternary carbonatoms, respectively (assignment by the DEPT-spectra).

Extraction and purification

Strain 553, The culture broth of strain 553 was filtered andthe filtrate (10 I) extracted with freshly distilled ethyl acetate(I I) to afford a crude extract (1'50 g). Methanolic extracts ofthe mycelium showed no biological activity. Three activecompounds with Rr 0'95,0'7 and 0'55, respectively (dichloro­methanej2% methanol as eluent) could be detected byspraying the tic autobiograms with the test organismCladosporium cucumerinum Ellis & Arthur (see below). Thecompounds also showed fluorescence on tic plates uponirradiation with 256 nm uv light. The crude extract wasdissolved in dichloromethane (10 ml) and applied to a3 x 60 cm silicagel (60 mesh, 200 g) column. Elution withdichloromethane (300 ml), dichloromethanejl % methanol(200 ml), and then dichloromethanej3% methanol (200 rnl)gave three fractions (the first 130 ml of dichloromethane werediscarded). The fractions were evaporated at reduced pressureand each fraction was purified separately by preparative tic(silica gel, I mm, 20 x 20 cm, dichloromethanejl % methanol).

Compound 1 was crystallized from n-hexane to afford37 mg of white needles (m.p. 50-51°); [aD25 = - 102(c 0.969, CHCl3); Anderson & Edwards (1983) m.p_ 51-52°;[a]D25 = -100 (c 1-0 in CHC13); ElMS (20°): mje (% ~.lse

peak) = 178 (100, M+), 160 (40), 149 (20), 134 (84), 104 (20),78 (20); ir (KBr): 2950 (OH-chelat.), 1650 (lactone), 1615,1460,1220,1210,1110,800 cm-\ uv (methanol): Amax (lge)211 (3'291), 246 (3'822), 313 (3'569); 1H-NMR (CDCl3):01'53 ppm (d, 1 = 6'3 Hz, 3 H, CH3CH-), 2-93 (d, 14 ,3 =

7'3 Hz, 2 H, 4-H), 4'73 (m, I H, 3-H), 6-69 (dd, 1ortho = 7'4 Hz,1meta = 0'8 Hz, I H), 6'89 (d, 1ortho = 8'1 Hz, I H, 6-H), 7'41(dd, 1ortho = 8'0 Hz, I Ht 11-04 (s, I H, OH); 13C-NMR(CDCI3): 020'75 ppm (p, C-3'), 34'56 (s, C-4), 76'11 (t, C-3),108'29 (g, C-4a), 116'18 (t), 117-92 (t), 136'13 (t, C-7), 139'41

(g, C-8a), 162'15 (g, C-8), 169'96 (g, C-l).Compound 2 was crystallized from dichloromethane j n­

hexane to afford 25 mg of white crystals (m.p. 145°; [alD25 =

-31 (c 1'85 ethanol); (Trofast & Wickberg, 1977) [alD25 =

+33 (c 1'33 in CHCl3); ElMS (110°): mje (% base peak) 284(I M+ + 2), 282 (I M+), 264 (100 M+ - H 20), 299(36M+-H20-CI), 201 (34), 173 (30), 156 (28),135 (24),100 (28), 65 (36), 43 (38); ir (KBr): 3440 (OH), 1716 (C = 0),1609 (C = C), 1570, 1395, 1377 cm-1; uv (ethanol): Amax (lge)208 (4-059), 232 (4'098), 301 (4'040); 1H-NMR (CDCl3):01'24 ppm (s, 3 H, CH3-C), 1'34 (s, 3 H, CH3-C), 1'60 (dd,1gem= 4-9 Hz, 110, 9 = 5-8 Hz, I H, 10-H), 1'91 (dd,1gem = 4-8 Hz,

110,9 = 8'3 Hz, I H, 10-H), 2-03 (d, 13',2' = 6'9 Hz, 3 H,3'-H),2'23 (dd, 19 ,10 = 5'9 and 8-3 Hz, I H, 9-H), 7-0 (g, 12',3' =

6'9 Hz, I H, 2'-H), 7'1 I (s, I H, 3-H); 13C-NMR (CDCI3):5 14'95 ppm (s, C-I0), 16'47 (p, C-3/), 25'00 (p, CH3C) 29-06(p, CH3C), 43-10 (g, C-I). 44'68 (t, C-9), 82-86 (g, C-8), 101'22(g, C-6), 127-26 (g, C-r'), 135-73 (t, C-2/), 137'35 (t, C-3),144'88 (g, C-4), 192'24 (g, C-5), 192'56 (g, C-2).

Compound .3 was isolated from the polar fraction.Evaporation of the solvent afforded 3 mg of crystals (m.p.149-155°, decomposition); ElMS (90°): mje (% base peak) =

Secondary metabolites of endophytic Pezicula species

260 (50 M+), 243 (20 M+-OH), 201 (100), 137 (20), 61 (56),43 (60); ir (KBr): 3102 (OH), 1638 (CHO), 1599, 1438,1310(C-O-), 1220, 1070 cm-l ; uv (methanol): "max (lge) 206(4'075), 234 (4'023), 278 (3'946), 314 (3'828); lH-NMR(COCla); 53'42 ppm (s, 3 H, CHaOCH2), 3'91 (s, 3 H, CHaO),4'72 (m, 2 H, -CH20), 6'49 (d, Jmeta = 2'2 Hz, I H, 3-H or5-H), 6'83 (m, I H, 3-H or 5-H), 10'38 (d, h: 6 = 0'8 Hz, I H,II-H); laC_NMR (COCla): 556'33 ppm (p, C-2 /), 58'73

(p, CHaOCH2), 73'2 (s, CH2), 97'80 (t), 106'35 (t), 146'62 (q),164'73 (q), 166'48 (q), 189'35 (q), 206 (t, C-1').

Strain 1156. Two different methods of culture wereinvestigated, both at 25° for 14 d in 5 % liquid biomaltmedium. Only one compound could be isolated from theshaken cultures. It was identified as (- )-mellein (compound1). The culture from a 5 I fermenter with constant pH andoxygen concentration contained two new biologically activecompounds with Rf 0'45-0'5 and 0'25-0'3 (dichloromethane),The crude extract (0'80 g) was separated as described forstrain 553 by a combination of column and thick layerchromatography on silica gel (eluents dichloromethane/O-3 %methanol).

Compound 4 was crystallized from dichloromethane/n­hexane (I: I) to afford 27 mg of white crystals (m.p. 130-132°);[a]D25 = + 130 (c 1'33 in CHCla) (McGahren, den Hende &

Mitscher, 1969; Strunz et al., 1969): m.p. 133-137° and[a]D25 = +129 (c 1'35 in CHCla); ElMS (20°): m/e (% basepeak) = 268 (4 M+ + 4), 266 (22 M+ + 2), 264 (34 M+),248/246 (10/15), 207/205 (64/100), 179/177 (52/82), 149(38), 113 (45); ir (KBr): 3400 (OH), 2950 (CHa), 1750 (C =0), 1730 (5-ring ketone), 1630, 1570, 1430, 1350, 1200,1030,960,820,730 em-I; uv (methanol): "max (lge) 290 (4'133); lH_NMR (COcla): 51-98 ppm (dd,Ja',2' = 6'9 Hz, Ja"l' = 1'5 Hz,3 H, 3'-H), 3'82 (s, 3 H, CHaO), 4'43 (s, I H, OH), 4'64 (5, I H,5-H), 6-48 (dq, Jtrans = 16'4 Hz, Jl',a' = 1'6 Hz, I H, II-H),6'78 (dq,Jtrans = 16'2 Hz,J2'a' = 6'9 Hz, I H, 2'-H); laC_NMR(COCla): 520-35 ppm (p, C-3'), 54'55 (p, CHaO), 65'27 (t, C-5),82-82 (q), 121'51 (t, C-2 /), 122-08 (q), 143-71 (t, C-1'),156-43 (q), 170'94 (q, CHaOC = 0), 187'83 (q, C-4).

Compound 5 was purified by two successive layerchromatographs (eluent dichloromethane) to yield 20 mg of anoil (Takei et al., 1980); m.p. 64-65°; ElMS (50°): m/e (%) =

183 (2 M+H+), 109 (92), 96 (100); ir (KBr): 2950 (CHa),1780 (y-Iactone), 1610, 1450, 1290, II00, 1050 em-I; uv(methanol): "max (lge) = 209 (3'776); lH-NMR (COcla):5 1'09 ppm (t, J = 7'4 Hz, 3 H, CHaCH2), 1'85 (m, 2 H,CH2CHa), 3'59 (ddt, Jaa.6a = 8'3 Hz, Jaa ,4 = 4'1 Hz, J =2'4 Hz, I H, 3a-H), 4-41 (dt, J4,aa = 3'9, J4 ,4' = 6'4 Hz, I H,4-H), 5'05 (d, ha, aa = 8'6 Hz, I H, 6a-H), 5'90 (d, Jgem =2'2 Hz, I H, 3'-H), 6'48 (d, Jgem = 2'2 Hz, I H, 3 /-H); laC­NMR (COCla): 59'09 ppm (p, CHa), 29'02 (s, CH2CHa), 43'69(t, C-4), 74'29 (t, C-3a), 86'33 (t, C-6a), 126'40 (s, C-3'), 134'62(q, C-3), 167'0 (q), 169'84 (q).

Strain 2386. The semi-solid agar culture of strain 2386 wasemulgated in a Braun mixer (type 4142), then extracted withfreshly distilled ethyl acetate affording a crude extract. Onefungicidal compound could be detected in an autobiogram(see below). The crude extract was separated as described for

1010

strain 553 by a combination of column and thick layerchromatography on silica gel (eluents: dichloromethane/2%methanol). The compound isolated was identical to compound4, which had been isolated from the culture broth of strainII56.

Comparative thin-layer chromatography

Cultures of strains 1917-1932 were macerated in a Braunmixer and then extracted with freshly distilled ethyl acetate.The residue was redissolved in methanol. Thin layerchromatograms of these extracts were developed togetherwith the reference substances mellein, cryptosporiopsin andmycorrhizin using 100 % dichloromethane as eluent. Fungicidalactivity of the substances was revealed in an autobiogram, forwhich the tIc was sprayed with a suspension of the testorganism C. cucumerinum in 2 % biomalt and incubated for2-3 d in a moist chamber at room temperature (Krohn et aI.,1992).

In a second experiment all 85 strains were extracted asabove. The eluent for the tIcs was methanol-dichloromethane,7: 93, again using mellein, cryptosporiopsin and mycorrhizinas references. The Revalues and colours revealed with uv at254 and 366 nm were used to evaluate the thin layerchromatograms.

RESULTS

In a preliminary test for the synthesis of fungicidal substances,confrontation tests were run between endophytic P. lividastrains (545, 554 and II56) and Fusicoccum galericulatum. Ascan be seen in Fig, I, strains Il56 and 554 of P. livida producesubstances which diffuse in the agar medium and inhibit thegrowth of F. galericulatum.

Screening

The screening for fungicidal, antibacterial and algicidalsubstances is presented for a selection of 19 strains of Pezicula.All tested strains of Pezicula exhibited fungicidal activity in theinitial screening tests (Table 2), whereby inhibition of Eurotiumrepens was most pronounced. All but strain 1927 inhibited E.repens with a zone of inhibiton of at least 10 mm, Antibacterialand algicidal activity was limited, only strains II56 and 553caused a marked inhibition of Chlorella. There were no speciesspecific differences with respect to the organisms inhibited bythe 19 strains tested. Because of their broad and strongantibiotic properties, strains 553 and II56 were chosen forfermentation and subsequent isolation of the active substances.As a result of the distinct tIc bands of its culture extracts, strain2386 was later included in this selection.

Structure determination

The structure of the unpolar compound 1 was elucidatedprimarily on the basis of NMR data, The three signals foraromatic protons at 86'69, 6'89 and 7'41 ppm and J = 8'0 Hzand J = 0-8 Hz are typical for ortho- and meta-couplings. Thesinglet at 5II'04 ppm is characteristic for a chelated hydroxyl

OH 0

Fig. 2. Compound 1, (R)-mellein.

B. Schulz and others 1011

Table 2. Biological activity of the Pezicula strains in screening

A Radius of zone of inhibition (mrn)

Bacteria Fungi Alga

Strain B.m. E.c. U.v. M.m. E.r. F.o. C.f.

553 3 0 12 7 32 5 10

1156 0 0 20 10 30 5 8

1917 1 1 2 2 28 0 5

1918 1 0 2 2 22 0 3

1919 1 1 3 2 25 0 3

1920 2 0 3 2 41 0 3

1921 3 0 1 0 18 0 0

1922 2 0 0 0 15 0 0

1923 0 0 1 0 10 0 1

1924 1 0 1 0 10 0 0

1925 5 I 3 1 23 0 1

1926 3 0 0 0 15 0 0

1927 1 2 2 2 4 0 2

1928 1 1 2 2 21 0 4

1929 0 0 0 0 21 0 1

1930 1 0 1 0 23 0 2

1931 1 0 0 0 22 0 0

B 1932 0 0 0 0 30 0 I

2386 7 1 3 1 27 0 1

Test organisms: B.m.. Bacillus megaterium; E.c.. Eschmchia coli; U.v.,llstilago violacea; M.m.. Mycofypha microspora; E.r.. Eurotium repens; F.o.,Fusarium orysporum; C.f.• Chlorella fusca.

The strains in bold print were used for subsequent fermentation.

Fig. 1. Confrontation tests of Pezicu/a /ivida (above) in (a) of strain554 and in (b) of strain 1156 against Fusicoccum ga/ericu/atum (below).

group and the doublet at 51'53 ppm for a methyl group in thevicinity of a CH-group. Based on these data, in combinationwith the melting point, optical rotation and molecular ion forCloHI003' the compound can be identified as (- )-mellein (1)(Fig. 2) (Anderson & Edwards, 1983). Mellein was first isolatedin 1933 from Aspergillus melJeus Yukawa (Nishikawa, 1933).The compound is identical with ochracin from Aspergillusochraceus K. Wilh. (Yabuta & Sumiki, 1933) and was alsoisolated from Lasiodiplodia theobromae (Pat.) Griffon & Maubl.and Septoria nodorum Berk. (Devys et al., 1980; Aldridge et al.,1971). The absolute configuration of the compound withnegative rotation was established to be (R) (Arakawa et al.,1969), but the antipode is also a natural product from FusariumIarvarum Fuckel and Cercospora spp. (Grove, 1972; Carmada,Merlini & Nasini, 1976; Grove, 1979).

The second compound from the fraction of mediumpolarity (Rr 0'7) showed peaks at m/e 282 and 284characteristic for the presence of one chlorine atom. The basepeaks at m/e 264/268 resulted by elimination of onemolecule of water. The IH-NMR showed three signals at51'24, 1'34 and 2'03 ppm, the latter coupling with an olefinicproton at 57'0. The signal for a second olefinic protonappears at 57'11 ppm and the signal groups at 51'91 (I H,dd) and 2'23 (I H, dd) are part of an AB-system that coupleswith a methine group at 51'60 ppm. The 13C-NMR spectrumshows signals for seven quaternary carbon atoms, the two at192-24 and 192'56 belonging to a carbonyl group. Thehighfield signal for a secondary carbon at 514'95 ppm couldbe part of a cyclopropyl ring. In combination with the datafrom the mass spectrum, the molecular formula C14H1S0 4CIcan be deduced and the compound identified as 6-hydroxy­8,8-dimethyl-4-(1-chlorprop-1-enyIHricyclo-[4.4.0.01-7-oxa­dec-3-en-2,5-dione. This compound is known as (+ )-mycor­rhizin A (Trofast & Wickberg, 1977) (for related structures seeChexal et al. (1979). However, an optical rotation of [aln

2S + 33in ethanol is recorded in the literature (Chexal et al., 1979),

Secondary metabolites of endophytic Pezicula species

Fig. 3. Three dimensional structure of compound 2,(- )-mycorrhizin A.

OR

eRO

Fig. 4. Compound 3, 2-methoxy-4-hydroxy-6-methoxymethyl­benzaldehyde.

whereas our compound reproducibly yielded values of[a]D2°-31 in ethanol.

To be entirely sure that our compound was in fact theantipode of the known (+ )-mycorrhizin A, an X-ray structureanalysis was performed confirming the absolute configurationof (- )-mycorrhizin A (2) (Fig. 3). Crystal data: C14Hl()Cl04,M r = 282'71, orthorhombic, P2I2I21, a = 8'010(3), b =

8'062(3), c = 20'544(7) A, V = 1326'7 N, Z = 4, (Mo Ka) =0'71073 A, T = - 100°C. Data collection: Yellow prism0'4 x 0'36 x 0'28 mm, Siemens R3 diffractometer, 3340 inten­sities (3061 unique), 28max 55°. Structure solution and refine­ment: Direct methods, refined on F2 (program SHELXL-93,G. M. Sheldrik, University of G6Hingen), H atoms withriding modeL wR(F2

) 0'109, R(F) 0'044, 176 parameters, 172restraints, 5 = 0'90, max. 0-21 e A-3. The absolute con­figuration was confirmed by an x refinement (Flack. 1983); xrefined to - 0'07 (10). Full details of the structure de­termination have been deposited at the Fachinformations­zentrum Karlsruhe, Gesellschaft fUr wissenschafHich-technischeInformation mbH, D-76344 Eggenstein-Leopoldshafen,Germany, from where this material can be obtained onquoting the full literature citation and the reference numberCSD 400974. Since then, (- )-mycorrhizin A (2) has also beenisolated from another of our endophytic fungi, Plectophomellasp. (C. Spory-Kliche & K. Krohn, unpublished).

Compound 3 (Fig. 4) showed a molecular ion at m/e 196and a fragment at 181 typical for loss of a methyl group. The

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o

Fig. 5. Compound 4 (+ )-cryptosporiopsin.

doublet at 510'38 is typical for an aldehyde proton couplingwith the aromatic proton at 6'83 ppm, confirmed by an ir bandat 1638 em-I. The meta-coupling of] = 2'2 Hz with a secondaromatic proton at 56'49 ppm defines the relative freepositions on the aromatic ring. The picture is completed bysignals for two methoxyl groups (53'91 ppm, aromatic,3'42 ppm non-aromatic) and a lowfield signal for a methylenegroup at 54'72 ppm. The I3C-NMR shows signals for twoprimary, one secondary and three tertiary carbon atoms.These data confirm the molecular formula ClOHI20 4. Therelative position of the substituents was determined bycomparison with synthetic material. The data of neither 2­methoxy-3-hydroxy-6-methoxymethyl-benzaldehyde (Saa etal., 1988) nor 5-methoxymethyl-o-vanillin (Rigler et a!., 1986)agree with those of the natural product. The oxygen functionortho to the aldehyde must be methylated because no lowfieldsignal for a chelated phenol can be detected in the IH NMRspectrum.

Compound 4 was isolated from the less polar fraction ofthe fermenter culture broth of P. livida (strain 1156). Peaks inthe mass spectrum at 264/266/268 (M+, M+ + 2 and M+ + 4)are characteristic for compounds with two chlorine atoms.Signals for two coupling olefinic protons (] = 16'4 Hz) at56-48 and 6'78 in the IH NMR spectrum indicate a trans­configuration. One proton (6'78 ppm) is further coupled(J = 6'9 Hz) with a methine proton and a long range coupling(J = 1'6 Hz) can be seen in the second signal at 6'48 ppm.The lowfield signal at 54'64 is probably attached to anelectronegative element such as chlorine; further signalsappear at 4'43 ppm (OH) and at 53'82 ppm (OCH3). The I3C_NMR shows signals for quaternary carbon atoms and a band at1730 cm-I in the ir spectrum could be assigned to anunsaturated keto group of a five-membered ring. The molecularformula CioHlOCl204 can be deduced from these data and thecompound is identified as methyl-3,5-dichloro-l-hydroxy­4-oxo-2-propenyl-2-cycIopenten- I -carboxylate, (+ )-crypto­sporiopsin (4) (Fig. 5) (McGahren, den Hende & Mitscher,1969; Strunz et al., 1969). The antipode (- )-cryptosporiopsinis also known as a natural product and was isolated fromPhialophora asteris (Dowson) Burge & I. Isaac (Lousberg, Tirilly& Schwab, 1976).

(+ )-Cryptosporiopsin was also the identification of thecompound with fungicidal activity isolated from P. cinnamomea(strain 2386) (Roemer, 1993).

The structure elucidation of compound 5, 4-epi-ethiosolide(Fig. 6), is described in a previous paper (Krohn et al., 1994).

B. Schulz and others

o

oFig. 6. Compound 5, 4-epi-ethiosolide.

Table 3. Biological activity of compounds isolated from P. livida in agardiffusion tests

Radius of zone of inhibition (mm)

Bacteria Fungi Alga

mg B.m. E.c. V.v. M.m. E.r. C.f.

(R)-mellein 1 1 0'9 2 2 8 0 6 14(R)( - )-mycorrhizin A2 1'2 8 3 14 10 14 3

(+ )-cryptosporiopsin 4 1'0 5 3 15 17 16 04-epi-ethiosolide 5 1'0 5 5 10 6 nt 3

Test organisms: B.m., Bacillus megaterium; E.c., Escherichia cali; V.v.,Ustilaga vialacea; M.m., Mycatypha micraspara; E.r. = Euratium repens; C.f. =Chiarella fusca. nt = not tested.

It was previously only known as a racemic synthetic compoundwith m.p. 64-65° (Birkinshaw, Oxford & Raistrick, 1936).

Biological activity

Compounds 1, 2, 4 & 5 were tested for antibacterial,fungicidal, algicidal (Table 3) and herbicidal activity. Therewas not an adequate amount of substance for testingcompound 3. All four compounds inhibited the fungal testorganisms. Only (R)-mellein inhibited the alga significantly(14 rom). In contrast to the screening results with strains 553,1156 and 2386, the pure compounds showed more anti-

1013

bacterial activity. All four compounds inhibited germinationof the two seedlings tested by 100 %, resulting in no growth.

Comparative thin-layer chromatography

Knowing that all 19 of the endophytic Pezicula strains selectedfor screening had produced biologically active substances inthe initial screening, we were interested to see if any or all ofthe compounds we had isolated from three of the strains werealso present in some of the other strains (1917-1932). It is alsopossible that one or more of the substances is typical for aparticular species of Pezicula. An autobiogram of extracts ofthese 16 cultures grown on 1% malt agar (36 d, roomtemperature) was run together with the reference substancesR-mellein, (+ )-cryptosporiopsin and (- )-mycorrhizin A (Fig.7). Cryptosporiopsin was detected in none of the extracts,mellein only in that of strain 1919. This means that either thesestrains do not produce mellein and cryptosporiopsin or not insufficient quantities to inhibit C. cucumerinum (test organismfor fungicidal activity) under these conditions. Mycorrhizin,judging from Revalues and inhibition of C. cucumerinum, waspresent in extracts of strains 1917-1920 and of strains1928-1932, meaning that all the tested strains of P. livida, P.sp. (strain 553) and all but one strain of P. carpinea (1927)synthesized (- )-mycorrhizin under these conditions of culture.Using a different medium (results not shown here) strain 1927also synthesized (- )-mycorrhizin. Mycorrhizin was detectedin none of the P. cinnamomea strains.

In a second experiment with 85 strains of Pezicula (P. livida,P. cinnamomea, P. carpinea, P. acericola, P. alnicola and P. sp.),including the 19 already tested in the autobiogram, theconditions of culture and eluent of the thin layer chroma­tograms were varied as described in Materials and Methods.Mellein could be detected under uv at 254 and 366 nm as ablue band with an Rr-value of 0'86 in all 85 strains.Cryptosporiopsin with an Revalue of 0'73 (black under uv at254 nm and light brown at 366 nm) was found in the extractsof strains 1156 (P. livida) and 2386 (P. cinnamomea) and in that

o R-melein

(+)-cryptosporiopsin

0 0 (-)-mycorrhizin

N N <"l '<t \Q r- oc 0\ 0 - NN N N U N N N N <"l <"l <"l

0\~ 0\ 0\ ~ ~ ~ 0\ 0\ ~ ~- - - ..; - -

Fig. 7. Autobiogram for fungicidal activity of culture extracts of Pezicula strains 1917-1932 with the reference compounds (r.c.)R-mellein, (+ )-cryptosporiopsin and (- )-mycorrhizin.

Secondary metabolites of endophytic Pezicula species

Table 4. Occurrence of Mellein, Mycorrhizin and Cryptosporiopsin in 85 Pezicula strains under two different conditions of culture

No. of strainstested Mellein Mycorrhizin Cryptosporiopsin

M MPY M MPY M MPY M MPY

P. acericola 0 I nt + nt ntP. alnicola 0 I nt + nt nt +P. carpinea 6 15 + +P. cinnamomea 5 16 + ±P.livida 8 27 + + ±P. spp. I 25 + ± ±

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Conditions of culture: M, 36 days on I % malt agar at room temperature; MPY, 28 days on MPY agar at room temperature; +, substance produced; -,substance not produced; ±, both strains that produced the substance and those that did not; nt, not tested.

of another strain of P. cinnamomea, in that of the P. alnicolastrain, and in that of two unidentified Pezicula species,including strain 553. Mycorrhizin was not detected in any ofthe extracts.

DISCUSSION

Our initial confrontation and screening tests showed that allthe Pezicula strains included in these studies synthesize, in vitro,fungicidal substances. The production of specific compoundsdepends on the conditions of culture, as has previously beenfound for the secondary metabolites of many fungi (Omura,1986). When a constant pH and a high level of oxygensaturation were maintained in a fermenter culture, strain 1156produced different substances than in shaken culture in whichthese parameters varied. Using solid media, all Pezicula strainssynthesized mellein growing on MPY-medium, but none onI % malt-medium. Additionally, we have shown that thesynthesis of certain metabolites may be specific for certainspecies and independent of the plant host. Mycorrhizin wassynthesized by P. livida and P. carpinea, but not by P.cinnamomea. Mellein was synthesized by all 85 Peziculaisolates included in these studies. This means that mellein maybe a compound characteristic for the genus and mycorrhizinone specific to certain species. The capability to synthesizecryptosporiopsin seems not to be characteristic of anyparticular species, but perhaps characteristic of some membersof the genus Pezicula and its anamorph Cryptosporiopsis.

The synthesis of cryptosporiopsin by six of the Peziculastrains is not surprising considering the fact that Crypto­sporiopsis is the anamorph state of Pezicula. Noble et al. (1991)found that endophytic Cryptosporiopsis sp. and Pezicula sp.isolated from Pinus sylvestris and Fagus sylvatica, respectively,both synthesize the same antifungal agent an echinocandin, invitro. Previously, cryptosporiopsin had been isolated from anunidentified species of Cryptosporiopsis (Stilwell. Wood &Strunz, 1969).

Mellein may be a substance responsible for the algicidalactivities of the Pezicula strains in the initial screening. In thetests for biological activity of the pure substances, 0'9 mg ofmellein caused a 14 mm zone of inhibition of the alga Chlorellafusca, in contrast to only 0-3 mm by 1·0 mg cryptosporiopsin,1'2 mg mycorrhizin A or 1'0 mg epi-ethiosolide.

Epi-ethiosolide, but especially mycorrhizin and crypto­sporiopsin, had good fungicidal properties in our tests for

biological activity. Mycorrhizin is known as a fungicidal agentof the tree root rot fungus, Heterobasidium annosum (Fr.) Bref.(Trofast & Wickberg, 1977) and cryptosporiopsin as anantifungal agent used in the protection of pinewood againstLenzites sepiaria (Wulfen ex Fr.) Fr. (Stillwell. Wood & Strunz,1969).

Assuming that the antifungal compounds we isolated fromPezicula are actually produced in nature, it is tempting tospeculate that the synthesis of these substances can be seen asPezicula's contribution to a mutualistic relationship with itshost. the fungicidal substances inhibiting parasites of the host.Certainly this explanation is too trivial. Moreover, it must bepostulated that Pezicula exerts the energy necessary tosynthesize these substances in order to inhibit the growth ofother fungi competing for the same environment. It is onlytolerated by its host because the infection remains verylimited until the host is stressed.

An example of how Pezicula species may be kept in thelatent phase by the host is demonstrated by the infection ofapples by P. malicorficis (H. S. Jacks.) Nannf. As long as theapple contains enough benzoic acid, the infection remainslatent. When the concentration of benzoic acid decreases asthe apple age, the latent infection of P. malicorficis developsinto bitter rot in stored fruits (Noble & Drysdale, 1983).

Endophytic fungi such as Pezicula, which are isolated froma very high percentage of apparently healthy tissues, arepresumably pathogens with low virulence. Sieber, Sieber­Canavesi & Dorworth (1991) postulate that the moreaggressive a pathogenic fungus is, the shorter its incubationperiod will be. Conversely, the less aggressive pathogens willhave a longer incubation period and be isolated from a higherpercentage of apparently healthy plant tissues, as is the casefor endophytic Pezicula species, which only become parasiticwhen their tree hosts are stressed (Kehr, 1992; Butin, 1989).During the long latency period, an endophytic fungusmaintains an equilibrium with its host. Finding that the fourantifungal compounds mycorrhizin, epi-ethiosolide, melleinand cryptosporiopsin are also such potent herbicides ispresumably significant. If these compounds are alsosynthesized in vivo, they may be important in maintaining anequilibrium during the latent phase and subsequently havepathogenic potential when the tree is stressed and the fungusbecomes parasitic.

We thank Bettina Riffel. Qunxiu Hu, Gabriele Giinther and

B. Schulz and others

Sebastian Swoboda for excellent technical assistance, DrsChristine Boyle, Rolf Kehr and Siegfried Draeger for fruitfuldiscussions and for critically reading the manuscript. We alsothank the Bundesministerium fur Forschung und Technik,BASF and the Fonds der Chemischen Industrie for financialassistance.

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