Mycopathologia 111: 9-15, 1990.

9 1990 Khlwer Academic Publishers. Printed h7 Belgium.

Antimicrobial activity of naphthoquinones from Fusaria

Robert A. Baker, ~ James H. Tatum ~ & Stan Nemec, Jr. 2

U,S. Citrus and Subtropical Products Laboratory, * Winter Haven, FL 33883-1909, USA;
2 U.S. Horticultural Research Station, 2120 Camden Road, Orlando, FL 32803, USA

Received 20 October 1989; accepted 27 November 1989

Abstract

Twenty-two naphthoquinone compounds isolated or derived synthetically from culture extracts of

Fusarium solani and F. oxysporum were examined for antimicrobial activity. Fifteen exhibited antibiotic
activity against Staphylococcus aureus, and 12 were active against Streptococcus pyogenes, but none were
active at the highest rate of 128 ~Lg/ml against Escherichia coli, Klebsiella pneumoniae, Salmonella typhi,
Proteus vulgaris, Serratia marcescens, or Pseudomonas aeruginosa. Of 8 plant pathogenic bacteria tested
against 11 naphthoquinones, Corynebacterium poinsettiae was inhibited by 6 compounds, and
Pseudomonas viridiflava was weakly inhibited by one. Only one of a group of 6 fluorescent soil
pseudomonads was inhibited by one naphthoquinone. Antifungal activity of 10 compounds against 8
fungal plant pathogens was limited to inhibition of Phytophthora parasitica by one naphthopyran.

Introduction in a competitive soil environment. Various formae

Naphthoquinone derivatives, both simple and
condensed, are produced by a number of Fusaria
[5, 20]. These secondary metabolites may be na-
tural products or compounds produced in re-
sponse to aging or other imposed physiological
stresses [ 13]. Their function is unclear; naphtho-
quinones of Fusarium solani (Mart) Appei. and
Wr. emend. Snyd. and Hans. have been impli-
cated in the manifestation of wilt in peas [9], but
their production was not correlated to pea stem
lesion pathogenicity [7].
In addition to phytotoxicity, several naphtho-
quinones exhibit antimicrobial activity against
bacteria, yeasts, and filamentous fungi [5, 9, 12].
The ability of F. solani to produce antimicrobial
compounds when stressed may be advantageous
speciales of F. solani and F. oxysporum Schlect.
emend. Snyd. and Hans. infect a number of hosts
to produce wilt and root rots, and various other
symptoms. In addition, nonspecialized forms of
these fungi are ubiquitous in many soils. In some
soils, aggressive colonization of root surfaces by
siderophore-producing pseudomonads are re-
ported to inhibit pathogenicity of Fusarium spp.
[10]. Production of antibiotic naphthoquinones
by Fusaria, if effective in inhibiting growth of
these pseudomonads, could confer a competitive
advantage to those Fusarium strains capable of
producing naphthoquinones with antibiotic prop-
erties.
Recently, as part of a study of their phyto-
toxicity, we isolated and identified 18 naphtho-
quinone derivatives from culture filtrates of

* South Atlantic, Agricultural Research Service, U.S. Department of Agriculture. Mention of a trademark or proprietary product
is for identification only and does not imply a warranty or guarantee of the product by the U.S. Department of Agriculture over
other products which may also be suitable.
10

F. solani [14, 15, 17] and 10 from culture filtrates
of F. oxysporum [ 15, 18, 19]. During structural
elucidations, 13 derivatives were also synthesized.
A number of these compounds and derivatives
had not previously been reported. Twenty-two of
these compounds were screened for antibiotic
activity against eight human bacterial pathogens;
the most active of these were tested fo'r antimicro-
bial activity against plant bacterial and fungal
pathogens and fluorescent soil pseudomonads.
Materials and methods
Naphthoquinone source
Isolates of F. solani and F. oxysporum were
obtained from fibrous roots of citrus trees by
culturing root steles on a modified Komada's
medium [ 11]. Cultures were single-spored, stored
at 27 ~ on V-8 agar slants and grown for
compound formation on glucose- or sucrose-
mineral salts media [4].
Table I.
Compounds 1, 2, 3, 9, 10, 11, 17, 19, and 20
(Table 1 and Figure 1) were produced in aerated
660 L tank cultures ofF. solani strains B3H, B3K,
and BP15-8 (NRRL 15980) using sucrose and
NH4NO 3 as the carbon and nitrogen sources [3].
Potential tank contamination was largely elimi-
nated by low culture pH (2.6). After 3-7 days,
the culture was f'dtered and naphthoquinones
were adsorbed from the filtrate on columns of
Amberlite XAD-7 resin. Compounds were eluted
with acetone and purified by silica gel column
chromatography and TLC [ 14, 16, 17].
Compounds 4, 8, 12, 13, 15, and 22 were puri-
fied by TLC from an ethyl acetate extract of an
F. oxysporum HC-7-1 (ATCC 64432) still culture
grown on glucose-mineral salts media, using
NaNO3 as the nitrogen source [15, 18].
Compounds 6, 18, and 21 were derived from still
cultures of F. solani [4], and compound 7 from
shake cultures ofF. solani [2]. Compound 5 was
synthesized from compound 4 by treatment with
methanolic HCI [15], and compounds 14 and 16
were derived from compounds 12 and 18 respec-
tively, by treatment with diazomethane [ 17].

Compound No Name

trans-Dihydrofusarubin
cis-Dihydrofusarubin
Fusarubin
9-0-Methylfusarubin
3-0, 9-0-Metylfusarubin
6 Anhydrofusarubin
7 Bostrycoidin
8 9-0-Methylbostrycoidin
9 Marticin
10 Isomarticin
11 2-Hydroxyjavanicin
12 8-0-Methyl-2-hydroxyj avanicin
13 8-0-Methyljavanicin
14 2-0, 8-0-Methyljavanicin
15 8-0-Methylsolaniol
16 5-0-Methyljavanicin
17 2,3-Dihydro-5,8-dihydroxy-6-methoxy-2-hydroxymethyl-3-(2-hydroxypropyl)- 1,4-naphthalenedione
18 Javanicin
19 5,8-Dihydroxy-2-methoxy-6-hydroxymethyl-7-(2-hydroxypropyl)-1,4-naphthalenedione
20 2•3-Dihydr•-5-hydr•xy-4-hydr•xymethy•-8-meth•xy-3-methy•naphth•(••2-b)furan-6•9-di•ne
21 Anhydrojavanicin
22 Nectriafurone
 11
o-H 0 H o~H 0 H

c , ~ o . . ,,~ . . ~ ~I I. 2 ~'~~"
0 O/RI""'-"4 O-X
H 0
CH30 H3
O--H 0 H O~H 0 H . ~ lJ %. . A -
c"3o o.
| 2 CH3
gO H.CH 3 H CH~H 22
R.a H-O 0 H-O
21 GHi ~ r

Fig. 1. Structure of naphthoquinone derivatives.

0t30 H3 CH30 CH3
0 H-O ' 0 H-O
RI R2 6 Assay procedures
3 H 0
4 H CH30
Both broth microdilution and agar diffusion as-
5 CH3 CH30 says were used to measure activities of
compounds. Compounds 1-22 were assayed
OR 0 0 H- 0 . H..../HX 0
'~ II C t
in vitro for activity in a broth microdilution assay
(National Committee for Clinical Laboratory
c .", o ~ ~ , , " T,+_ IT "'T ' ~7':~
%o~,,?.J~'<7, Standards) against Staphylococcus aureus Rosen-
O--H 0 a r H,~-,H 3
0 H--O bach (Smith), Streptococcus pyogenes Rosenbach
7 R=H 9 C-12 = 5
8 R=CH5 I0 C-12=R
(4), Escherichia coli (Migula) Castellani and
Chalmers (257), Klebsiella pneumoniae A.
Schroeter, Salmonella typhi (Schroeter) Warren
and Scott (P58A), Proteus vulgaris Hauser (100),
Serratia marcescens Bizio (SM), and Pseu-
Re o domonas aeruginosa (Schroeter) Migula (B).
R S ~ RI Compounds were dissolved in either ethanol or
dimethyl sulfoxide for incorporation in the assay.
The minimum inhibitory concentration (MIC)
was determined as the lowest level of compound
RI R2 R3 R4 R5 R6
at which culture turbidity failed to develop.
0
II OH CHz-C-CH 3 OH CHsO H OH
Antibacterial activities of compounds 1, 2, 6, 7,
0
9, 13, 15, 17, 19, 20, and 21 were measured with
12 OH CH2-C-CH 3 OH CH30 H CH30 the agar diffusion method against the following
0 plant pathogenic bacteria: Pseudomonas cepacia
II
13 CH3 CH2-C-GH 3 OH CH30 H CH30 Palleroni & Holmes (609), P. cichorii (Swingle)
0 Stapp (5359), P. syringae pv. phaseolicola (Burk-
14 CH30 CH?.-~-CH 3 OH CH30 H CH30 holder) Young (819), P. viridiflava (Burkholder)
I
OH Dowson (5777-4), P.fluorescens (Trevisan)
15 CH3 CHz-CH-CH3 OH CH30 H CH30
Migula (F35RT), Xanthomonas campestris pv.
Ii
0 vesicatoria (Doidge) Dye (83-38), X. campestris
16 CH30 H CH30 CH3 CH2-C-CH 3 OH
campestris (Pammel) Dowson (FTCC 081-2387),
OH
17 H , CH20H H, CH2-CH - CH3 OH CH30 H OH
and Corvnebacterium poinsettiae (Hedges)
Dowson (ATCC 9682) (Curtobacteriumflaccum-
RI Rz
faciens). These compounds were also tested
0
0i H--O against a group of six unspeciated fluorescent
ii H ~ RI
18 CH3 CH2-G-CH 3 pseudomonads isolated from citrus grove soils on
S-1 medium [6]. For assay purposes, all plant
19 CH20 H CH2-CH.CH 3 0 H--O pathogenic bacteria and soil pseudomonads were
12

streaked on King's B medium plates; P. viridiflava Table2. Antibacterial activity of naphthoquinone deriva-
tives against Staphylococcus aureus and Streptococcus
and soil pseudomonads were also assayed on S-1
pyogenes.
and D-4 media [8]. Plates were surface dried
before inoculation and held 3-6 hours after inocu- Compound No MIC (gg ml)
lation before applying compounds in impregnated
discs or liquid wells. S. aureus S. pyogenes
Impregnated discs were prepared by dissolving
1 0.5 16
compounds in acetone, applying to 14mm 2 I 32
Whatman No. 1 filter paper discs at 20, 40, and 3 16 > 128
80 pg levels, and air drying. Four discs were 4 > 128 > 128
applied per plate, and zones of inhibition were 5 2 > 128
6 2 8
measured after 24 hours at 27 ~C. Compounds 2
7 16 16
and 9, which are more soluble in water, were also 8 > 128 > 128
tested as aqueous solutions added to 10 m m wells 9 128 128
aseptically cut in the streaked plate. Four wells 10 > 128 128
were cut per plate, and levels of 12.5, 25, and 11 > 128 128
50 #g well were added in 0.1 mL volumes. 12 > 128 > 128
13 4 > 128
Compounds 1, 2, 6, 7, 13, 15, 17, 19, 20, and 21 14 > 128 > 128
were also tested for activity against Phomopsis citri 15 32 64
Fawc. (N-417), Colletotrichum gloeosporioides 16 4 > 128
(Penzig) Sacc. (N-622), Phytophthora parasitica 17 1 0.5
(Dastur) (N-107), Diplodia natalensis P. Evans 18 0.5 > 128
19 1 64
(N-384), Alternaria cirri Ellis and Pierce (N-251), 20 4 64
Geotrichum candidum Link (N-66), F. solani 21 0.25 > 128
(NRRL 15980) and F. oxysporum (ATCC 64432). 22 > 128 128
Four 14 m m discs impregnated with 20/~g of
compound were applied per plate to one day old hydroxyl or quinone function access to the 4 or 8
cultures on PDA (lima bean agar for Phytophtho- position for hydrogen bonding. Methyl esterifi-
ra), and zones of inhibition were measured after cation of a ring hydroxyl (as in compounds 13, 14,
three days at 27 ~ 15, or 16) reduces activity. If hydrogen bonding of
the oxopropyl ketone to the ring hydroxyl at the
Results and discussion
4 position is blocked (as in compound 11, where
a hydroxyl at the 2 position would hydrogen bond
Eleven of the 22 compounds tested by micro- the ketone), activity is greatly reduced.
dilution assay were active against Staphylococcus Compounds 1 and 2, which are stereoisomeric
aureus at MIC values < 4 gg mL (Table 2). Of naphthopyrans, also exhibit strong activity
these, nine appear to have similarities of configu- against S. aureus. However, the pyran ring of
ration which correlate to activity. Five these compounds opens readily in aqueous
(compounds 13, 16, 17, 18, and 19) are naphtho- solution to yield naphthoquinone structures equi-
quinones with a 2-oxopropyl or 2-hydroxypropyl valent to compound 17. Compounds 20 and 21,
group at the 3 or 7 position. The ketone or which are also quite active against S. aureus, are
hydroxyl of this group is free to hydrogen bond to naphthofurans. In these compounds, the hy-
the adjacent hydroxyl or quinone function of the drogen bonding between a hydroxyl on a propyl
naphthoquinone nucleus at position 4 or 8. group to the ring oxygen has been replaced by the
The propyl group can contain either a ketone or ether linkage of the furan. Activity exhibited by
hydroxyl function, since enolization of the compounds 5 and 6, both fusarubin derivatives, is
naphthoquinone would allow the corresponding not readily correlated to their structures.
 13

Streptococcus pyogenes, the other gram-positive
human pathogen tested, was generally less sensi-
tive to this series of compounds. Only one
compound, 17, was active at an MIC of
< 4 #g mL. Javanicin and javanicin derivatives
(compounds 13, 16, 18, and 21), which were quite
active against S. aureus, were inactive against
S. pyogenes. None of the 22 compounds exhibited
any activity at 128 ~g mL against the six gram-
negative human pathogens tested (data not
shown).
Few of these naphthoquinones have been pre-
viously examined for antibiotic activity. Of those
tested, most have exhibited activity against gram-
positive bacteria, with MIC values from 2 to
500 ~tg mL (Table 3). A comparison of published
MIC values for S. aureus to those of the present
study showed our culture was less sensitive to
compound 7 (bostrycoidin), but more sensitive to
compounds 6 and 18 (anhydrofusarubin and
javanicin) [ 1, 5].
Ammar et al. [1] reported MIC values of 75
and 100 ~tg mL for anhydrofusarubin against the
gram-negative bacteria E. coil and Serratia mar-
cescens, respectively. In our study, neither of these
organisms were inhibited by 128 #g mL of this
compound. Using discs containing 20/~g of
material, McColloch et aL [ 12] found
compounds 1, 2, 3, 6, and 18 were inactive against
E. coli and Pseudomonas fluorescens; Proteus vul-
garis gave a weak response only to compound 2.
In our broth microdilution assay, we found
P. vulgaris to be insensitive to compound 2 at
128 ~tg mL. Such variations in MIC values may
be due to differing assay procedures or to different
sensitivities of individual cultural strains.
Of 11 compounds assayed for antibiotic acti-
vity against eight gram-negative plant pathogens,
only one, compound 17, caused weak inhibition.
This compound was slightly active against
Pseudomonas viridiflava at 80 ~g disc, and only
when tested on S-1 medium. When this same
group of compounds was tested against six un-
speciated fluorescent soil pseudomonads, only
one isolate was weakly inhibited, and only by
compound 17 at 80 #g disc on S-1 medium (data
not shown).
Corynebacterium poinsettiae, a gram-positive
plant pathogen, was much more sensitive; six of
the compounds (1, 2, 6, 17, 19, and 20) produced
zones of inhibition on inoculated King's B
medium plates (Table 4). Against this organism,
the most effective antibiotics were compounds 20
and 17, two newly described naphthoquinones
from F. solani [16].
These results, together with the data on activity

Table 3. Reported MIC values or antibiotic activity of naphthoquinones against gram-positivebacteria.

Organism MIC values (~tgml) Reference

compound

I 2 3 6 7 9 18

Staphylococcus aureus 2 20 5
S. aureus 50 1
Bacillus subtilis 2.0 2 500 20 5
B. subtilis 20 60 20 9
B. subtilis 40 I
S. aureus 0.5 1 16 2 16 128 0.5 this paper

Antibiotic activity ( + or - ) of 20 #g discs

B. subtilis + + + + + 12
Streptococcus faecalis + + + + + 12
Streptomyces albus + + - _ 12
14
Table 4. Inhibition of Corynebacterium poflTsettiae growth on
King's B medium by naphthoquinone impregnated discs.
Values are inhibition zone diameter -disc, in mm.*
Conc. Compound
( t~g disc)
1 2 6 17 19 20
20 2.5 0 0 3.8 1.8 7.0
40 6.0 1.2 0 9.8 3.2 11.0
80 8.0 5.5 2.2 14.2 2.2 18.0
* Compounds applied in acetone to 14 mm Whatman No. I
filter paper discs, dried, discs placed four per plate 3-6 hours
after inoculation, and inhibition zones measured 24 hours
later.
against human bacterial pathogens, show this
series of c o m p o u n d s to be relatively ineffective
against gram-negative bacteria. This is consistent
with the literature on those few c o m p o u n d s pre-
viously examined for antibiotic activity. In light o f
the low activity expressed against gram-negative
fluorescent soil pseudomonads, it is unlikely that
these c o m p o u n d s would function effectively as
inhibitors o f p s e u d o m o n a d growth in Fusarium-
suppressive soils.
Ten of these c o m p o u n d s applied as 20 #g discs
to P D A plates inoculated with eight citrus fungal
pathogens yielded only one positive response.
Phytophthora parasitica was the only pathogen
affected, and only by c o m p o u n d 2, cis-dihydro-
fusarubin (9.2 mm average zone of inhibition).
Few naphthoquinones have been reported to have
antifungal activity. Botrytis allii spore germination
was inhibited by novarubin and norjavanicin at 5
and 10/~g m L respectively [9]. Neither of these
naphthoquinones were produced by the Fusaria
used in this study. Both c o m p o u n d s lack a sub-
stituent group at the 2 position adjacent to a
propyl group, a feature not shared by any of the
c o m p o u n d s we tested. Candida albicans was
inhibited by 20 gg discs of c o m p o u n d s 1, 2, 7, and
norjavanicin, and Saccharomyces cerevisiae was
inhibited by c o m p o u n d s 2, 3, and 6, suggesting
that yeasts may be more sensitive than fila-
mentous fungi to naphthoquinones [12].
In general, our results confirm earlier, more
limited studies on individual c o m p o u n d s in this
series o f n a p h t h o q u i n o n e s . Activity against gram-
negative bacteria was negligible; only two in-
stances of weak inhibition were seen. Those
gram-positive bacteria tested were much more
sensitive, with 11 of the c o m p o u n d s inhibiting
S. aureus at M I C values of 4/.tg m L or less. Only
one c o m p o u n d exhibited any activity against a
group of filamentous fungi, and only against Phy-
tophthora. These results suggest this series of
naphthoquinones would offer little protection to
Fusarium against most other soil microorganisms.
Acknowledgements
The authors wish to thank Dr. Roy Cleeland of
H o f f m a n - L a R o c h e , Inc., Nutley, N e w Jersey, for
performing the broth microdilution assays, and
Ms. Stephanie Burgess, Florida D e p a r t m e n t o f
Agriculture, Gainesville, Florida, for bacterial iso-
lates.
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