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Antifungal activity of isoflavonoids in different reduced stages on Rhizoctonia solani and Sclerotium rolfsii
Phytochemistry,Vol. 29, No. 3, pp. 801-803, 1990.
003I 9422/90$3.00+ 0.00 Pergamon Press plc
Printed in Great Britain.
ANTIFUNGAL ACTIVITY OF ISOFLAVONOIDS IN DIFFERENT REDUCED STAGESON RHIZOCTONIASOLANI AND SCLEROTIUMROLFSII MARTIN WEIDENB~RNER, HOLGER HINDORF, HEM CHANDRA JHA,* PRODROMOS TSOTSONOS* and HEINZ EGGE* Institut fiir Pflanzenkrankheiten
der Universitgt Bonn Nussallee 9, D-5300 Bonn 1, F.R.G.; Institut fiir Physiologische Chemie der Universitlt Bonn Nussallee 1l,* D-5300 Bonn 1, F.R.G. (Received in revised form 21 August 1989)
Key Word Index-Rhizcotonia relationships.
solani; Sclerorium ro(fsii; isoflavonoids;
antifungal activity; structure-activity
Abstract-Two naturally occurring isoflavones, genistein and biochanin A, and their dihydroderivates (isoflavanones) as well as nine perhydrogenated isoflavones (isoflavans) were tested for their effects on mycelial growth of the two soil borne fungi Rhizoctonia solani and Sclerotium rolfsii. All the isoflavonoids of the biochanin A series showed high antifungal activity. Genistein isoflavan and the other isoflavans with two hydroxyl groups and one methoxy group were fungitoxic, while isoflavans with two or three methoxy groups were almost inactive.
INTRODUCTION The effects of isoflavonoids on microorganisms have been investigated by several groups of workers [l-5]. It has been established that some of them possess strong antimicrobial activities [6-91. However, less information is available regarding the structure-activity relationship of these compounds. In continuation of our previous work [l, 6, 10, 111 further data are presented in this publication. RESULTS AND DISCUSSION In the A,_, series the isoflavan A, was the most effective substance inhibiting mycelial growth of R. solani to 79.5% and S. rolfsii to 91.6% at a concentration of 0.8 mM. In contrast to the A, _ 3 series, in the B, _ 3 series all the isoflavdnoids showed high antifungal activity. At the highest concentration tested (0.8 mM) these isoflavonoids inhibited the growth of R. solani and S. rolfsii in the range of 7&80% and about 90% respectively. The antifungal activity of the isoflavan C, at concentrations of 0.2 and 0.8 mM was also high. R. solani was inhibited up to 80% and S. rolfsii to 91% at both these concentrations. The isoflavans D, and E, caused significant inhibitions of mycelial growth only in the highest concentration (0.8 mM). The inhibitory effects of both towards S. rolfsii amounted to 94%. The other four isoflavans, F,-I,, possessed low antifungal activity. A maximum inhibition of S. rolfsii up to 57.9% at a concentration of 0.8 mM was observed only in the case of the isoflavan I,. The isoflavan H, stimulated mycelial growth of S. rolfsii at concentrations of 0.05 and 0.2 mM to 23%, while there was no significant activity at a concentration of 0.8 mM. As in the case of 5,7,4’-trihydroxy-isoflavan A, against R. solnni and S. rolfsii in the present investigation, Adesanya et al. [12] found that the 7,2’,4’-trihydroxyisoflavan demethylvestitol was fungitoxic to Cladosporium cucumerinum and Aspergillus niger. However, in our 801
previous investigations [ 1, 1 I] the 6,7,4’-trihydroxy-isoflavan was inactive against different fungi. Also the 6,7,4’trihydroxy-isoflavone/isoflavanone showed low activity while the corresponding substances of the genistein series A, and A, were active to some extent. Similar results were obtained by Adesanya et al. [12] with genistein. The differences in activity between the 6,7,4’-trihydroxyisoflavonejisoflavanone and the substances A, and A, may be ascribed to the presence of the hydroxyl group at C-5 in the latter compounds. In other investigations [l 1, 13, 141, it was found that even a smaller number of hydroxyl groups can confer antifungal activity to isoflavans. This shows that the degree of reduction, the position and the number of hydroxyl groups together constitute important parameters for high activity of the substances. It was shown earlier [6] that unsubstituted isoflavonoids (isoflavone, isoflavanone, isoflavan) possessed weak antifungal activity and only after introducing suitable substituents in particular positions were these isoflavonoid structures fungitoxic. In our present investigation, we find that the substituent pattern of B,_, series, viz. two hydroxyl groups at C-5 and C-7 and one methoxy group at C-4’, is conducive to a high antifungal activity. In this case the degree of reduction seems to play only a minor role. Among the tested substances, a comparison of the effectiveness of the isoflavones genistein (A,) and biochanin A (B,) reveals that the presence of a methoxy group instead of the hydroxyl group at C-4’ bestows a high activity. Johnson et al. [15] obtained similar results when testing these two substances against Cercospora beticola and Monilia fructicola. Furthermore, the isoflavone B, is even more active than the isoflavan B,. This is in contrast to the hypothesis that a skewed, aplanar molecular shape of the isoflavans [16,17] is essential for a high activity [8]. In’one of our investigations [6],it was found that the 6,7-dihydroxy-4’-methoxy-isoflavan was fungitoxic to different moulds of the genus Aspergillus. These results led to the conclusion that two hydroxyl
M. WEIDENB~RNER et al.
802
the activity. Also in the present investigation, the isoflavans F,, G,, H, and I, with two or three methoxy groups were almost inactive. However, sativan, a 7hydroxy-2’,4’-dimethoxy-isoflavan was found to be more active than the corresponding 7,2’-dihydroxy-4’methoxy-isoflavan vestitol [16]. In a recent investigation [Ill, it was found that the 6,7-dihydroxy-Y-methyiisoflavan is highly active.
Aa”,
A,Bl
A,A, R' = R' = R' = OH.R) = H B,Bz
R’ =
OMe,
R2 =
OH, R’ =’ H
R’=
EXPERIMENTAL
A3
R’ =
R’
R4 =
OH
M,
R’=
OMc,
R’=
R’=
CI R’=
OMe.
R’ =
H. R’ =
0,
R’ =
R’=
OH,R’=
H.R’=
OMe
tr
R’ =
R3 =
OH.
H. R’ =
OMe
F,
R’=
R’ =
OMe.
R’ =
H. R’ =
OH
(13
R’ =
R’=
OMe.
R’=
H.R’=
Ott
n,
R’ =
OH, R’ =
R’=
R3 =
I3
=
R’
R’ =
=
f,
OH, R3 = R’ =
H Ott
ti, R’ = R4 = OMe
R’ =
OMe.
R’ =
H
and one methoxy group in an isoflavan guarantee a high activity irrespective of the position of the substituents in the molecule, provided one of the rings is disgroups
ubstituted. This is demonstrated by the isoflavans B,, C,, D, and E,, which have two hydroxyl groups and one methoxy group in their molecules and whose ring A is
disubstituted. The importance of derivatising phenolic groups to ethers for increased activity was established by Van Etten [16], Ingham [18], Ingham [19] and Matthews and Van Etten [20]. The presence of an ether function apparently protects the substance against microbial transformations [21]. It was also shown [14, 163 that two hydroxyl groups and one substituted oxygen function other than a methyl-ether group make the isoflavan strongly antifungal. Further methylation could impair
Table
1. Effect of isoflavonoids
on mycelial growth
Isoflaronoids. 13 isoflavonoids were tested in 3 concentrations for their effect on mycelial growth of two soil-borne fungi, R. solani and 8. ro&i. Three compounds belong to the 5,7,4’trihydroxyisoflavone series A, _3, while 5,7-dihydroxy-4’-methoxy-isoflavonoids belong to the series B,-3. The isoflavonoids of each series differ only in the degree of reduction. Besides the isoflavans A, and B, seven other isoflavans C,-I, were also tested (Table 1). In order to study the influence of gradual reduction on the antifungal activity, the isoflavones were catalytically (Pd-C Hz) reduced to isoflavanones and isoflavans. The purity and authenticity of these products were verified by physical methods (NMR and MS). All the substances were synthesized in the Institute of Physiological Chemistry of the University of Bonn. Fungi and culfure. The soil-borne fungi R. .solani and S. rolfsii were originally isolated from Phuseolus ou!yaris and Iris holhdim in German soils near Cologne and kept in the fungus collection of the Institut fur Pflanzenkrankheiten at Bonn. Both fungi are pathogenic to soybeans 1221. The effect of isoflavonoids on my&al growth was investigated in liquid culture. The soln contained 30 g malt extract and 3 g peptone per 1 I distilled H,O. Me&O served as the solvent for the addition of isoflavonoids. The solvent concentration in the soln was maintained at 1.1%. Medium (20ml) including the particular isoflavonoid in the required concentration was transferred to 100 ml Erlenmeyer flasks and inoculated with five small pieces of mycelium (5 mm in diameter). The flasks were incubated at 23-25” on a reciprocal shaker for 7 days. Concentrations of isoJ7avonoid.s. From phytoalexin experiments [l, 231, it is known that concn in the range of 10-5-10-3 M exhibit fungal inhibition. Therefore the concentrations of 0.5, 2.0 and 8.0 x lo-“ M respectively were used in these tests.
of R. solani and S. rolfsii (% difference in wt as compared S. rolfii
R. solani Compound
0.05 mM
A, - 25.0
A, A, B, B, B, C, D, E, F, G, H, I,
-20.6 - 8.7 - 7.9 - 29.9 - 19.7 - 22.9 [( +)indicates
significant
growth
0.2 mM
0.8 mM
-33.5 -16.8 +31.6 -50.1 - 73.4 -40.5 - 79.9 - 10.1 - 16.9 -31.7 - 35.2 -25.7 - 14.6
- 50.0 -79.5 - 79.6 -71.1 - 76.6 - 78.3 - 86.3 - 84.4 - 39.4 -24.2 - 23.5 - 15.7
stimulation,
with the control)
(-)
indicates
significant
0.05 mM -44.5 - 52.4 + 27.7 - 65.4 -26.5 -28.8
f22.8 - 17.5 growth
inhibition].
0.2 mM
0.8 mM
- 64.6 - 58.7
- 66.6 -63.0 -91.6 -90.8 -92.1 - 89.2 -90.8 -94.3 -93.7
‘- 57.0 -92.3 -49.3 -90.9 - 12.5 -23.7 - 36.5 + 20.6 -22.9
- 38.2 - 57.9
Antifungal
activity
Evaluations ofresults. Round filter papers (Schleicher & Schiill No. 595) were weighed after drying for 24 hr at 105”. After filtration of the culture, the residue was dried and weighed as described above. The difference of the weighings gave the dry wt of the fungus. The mean value ofeight repetitions for each concentration and fungus was used for calculation. The data were evaluated by analysis of variance. Probability of single differences was calculated at the 5% level. REFERENCES
I. Kramer, R. P., Hindorf, H., Jha, H. C., Kallage, J. and Zilliken, F. (1984) Phytochemistry 23, 2203. 2. Cruickshank, I. A. M. (,l962) Aust. J. Biol. Sci. 15, 147. 3. Smith, D. A. (1976) Physiol. Plant Pathol. 9, 45. 4. Van Etten, H. D. and Stein, J. I. (1987) Phytopathology 68, 1276. 5. Wyman, J. G. and Van Etten, H. D. (1978) Phytopathology 68, 583. 6. Weidenbiirner, M., Jha, H. C., Hindorf, H., Weltzien, H. C. and Zilliken, F. (1987) Med. Fat. Landbouww. Rijksuniu. Gent. 52, 933. 7. Skipp, R. A. and Bailey, J. A. (1977) Physiol. Plant Pathol. 11, 101. 8. Perrin, D. R. and Cruickshank, I. A. M. (1969) Phytochemistry 8, 971.
of isoflavonoids
803
S. S. and Smith, D. A. (1980) Phytopathol9. Gnanamanickam, ogy 70,894. M., Jha, H. C. and Hindorf, H. (1988) Med. IO. Weidenbiirner, Fat. Landbouww. Rijksuniv. Gent. 53, 1417. M., Hindorf, H., Jha, H. C., Tsotsonos, P. 11. Weidenbiirner, and Egge, H. (1989) Phytochemistry 28, (in press). 12. Adesanya, S. A., O’Neill, M. J. and Roberts, M. F. (1986) Physiol. Plant Pathol. 29, 95. 13. O’Neill, T. M. and Mansfield, J. W. (1982) Trans. Br. Mycol. Sot. 79, 229. 14. Stossel, P. (1985) Physiol. PIant Pathol. 26, 269. 15. Johnson, G., Maag, D. D., Johnson, D. K. and Thomas, R. D. (1976) Physiol. Plant Pathol. 8, 225. 16. Van Etten, H. D. (1976) Phytochemistry 15, 655. 17. Van Etten, H. D. and Pueppke, S. Cl. (1976) in Biochemical Aspects of Plant-Parasite Relationship (Friend, J. and Threlfall, D. R., eds) Ann. Proc. Phytochem. Sot. 13. 18. Ingham, J. L. (1976) Phytochemistry 15, 1489. 19. Ingham, J. L. (1977) Phytochemistry 16, 1279. 20. Matthews, D. E. and Van Etten, H. D. (1983) Arch. Eiochem. Biophys. 224, 494. 21. Sharma, R. D. (1979) Lipids 14, 535. 22. Sinclair, J. B. (1982) Compendium of Soybean Diseases 23. Van Etten, H. D. and Bateman, D. F. (1971) Phytopathology 61. 1363.
003I 9422/90$3.00+ 0.00 Pergamon Press plc
Printed in Great Britain.
ANTIFUNGAL ACTIVITY OF ISOFLAVONOIDS IN DIFFERENT REDUCED STAGESON RHIZOCTONIASOLANI AND SCLEROTIUMROLFSII MARTIN WEIDENB~RNER, HOLGER HINDORF, HEM CHANDRA JHA,* PRODROMOS TSOTSONOS* and HEINZ EGGE* Institut fiir Pflanzenkrankheiten
der Universitgt Bonn Nussallee 9, D-5300 Bonn 1, F.R.G.; Institut fiir Physiologische Chemie der Universitlt Bonn Nussallee 1l,* D-5300 Bonn 1, F.R.G. (Received in revised form 21 August 1989)
Key Word Index-Rhizcotonia relationships.
solani; Sclerorium ro(fsii; isoflavonoids;
antifungal activity; structure-activity
Abstract-Two naturally occurring isoflavones, genistein and biochanin A, and their dihydroderivates (isoflavanones) as well as nine perhydrogenated isoflavones (isoflavans) were tested for their effects on mycelial growth of the two soil borne fungi Rhizoctonia solani and Sclerotium rolfsii. All the isoflavonoids of the biochanin A series showed high antifungal activity. Genistein isoflavan and the other isoflavans with two hydroxyl groups and one methoxy group were fungitoxic, while isoflavans with two or three methoxy groups were almost inactive.
INTRODUCTION The effects of isoflavonoids on microorganisms have been investigated by several groups of workers [l-5]. It has been established that some of them possess strong antimicrobial activities [6-91. However, less information is available regarding the structure-activity relationship of these compounds. In continuation of our previous work [l, 6, 10, 111 further data are presented in this publication. RESULTS AND DISCUSSION In the A,_, series the isoflavan A, was the most effective substance inhibiting mycelial growth of R. solani to 79.5% and S. rolfsii to 91.6% at a concentration of 0.8 mM. In contrast to the A, _ 3 series, in the B, _ 3 series all the isoflavdnoids showed high antifungal activity. At the highest concentration tested (0.8 mM) these isoflavonoids inhibited the growth of R. solani and S. rolfsii in the range of 7&80% and about 90% respectively. The antifungal activity of the isoflavan C, at concentrations of 0.2 and 0.8 mM was also high. R. solani was inhibited up to 80% and S. rolfsii to 91% at both these concentrations. The isoflavans D, and E, caused significant inhibitions of mycelial growth only in the highest concentration (0.8 mM). The inhibitory effects of both towards S. rolfsii amounted to 94%. The other four isoflavans, F,-I,, possessed low antifungal activity. A maximum inhibition of S. rolfsii up to 57.9% at a concentration of 0.8 mM was observed only in the case of the isoflavan I,. The isoflavan H, stimulated mycelial growth of S. rolfsii at concentrations of 0.05 and 0.2 mM to 23%, while there was no significant activity at a concentration of 0.8 mM. As in the case of 5,7,4’-trihydroxy-isoflavan A, against R. solnni and S. rolfsii in the present investigation, Adesanya et al. [12] found that the 7,2’,4’-trihydroxyisoflavan demethylvestitol was fungitoxic to Cladosporium cucumerinum and Aspergillus niger. However, in our 801
previous investigations [ 1, 1 I] the 6,7,4’-trihydroxy-isoflavan was inactive against different fungi. Also the 6,7,4’trihydroxy-isoflavone/isoflavanone showed low activity while the corresponding substances of the genistein series A, and A, were active to some extent. Similar results were obtained by Adesanya et al. [12] with genistein. The differences in activity between the 6,7,4’-trihydroxyisoflavonejisoflavanone and the substances A, and A, may be ascribed to the presence of the hydroxyl group at C-5 in the latter compounds. In other investigations [l 1, 13, 141, it was found that even a smaller number of hydroxyl groups can confer antifungal activity to isoflavans. This shows that the degree of reduction, the position and the number of hydroxyl groups together constitute important parameters for high activity of the substances. It was shown earlier [6] that unsubstituted isoflavonoids (isoflavone, isoflavanone, isoflavan) possessed weak antifungal activity and only after introducing suitable substituents in particular positions were these isoflavonoid structures fungitoxic. In our present investigation, we find that the substituent pattern of B,_, series, viz. two hydroxyl groups at C-5 and C-7 and one methoxy group at C-4’, is conducive to a high antifungal activity. In this case the degree of reduction seems to play only a minor role. Among the tested substances, a comparison of the effectiveness of the isoflavones genistein (A,) and biochanin A (B,) reveals that the presence of a methoxy group instead of the hydroxyl group at C-4’ bestows a high activity. Johnson et al. [15] obtained similar results when testing these two substances against Cercospora beticola and Monilia fructicola. Furthermore, the isoflavone B, is even more active than the isoflavan B,. This is in contrast to the hypothesis that a skewed, aplanar molecular shape of the isoflavans [16,17] is essential for a high activity [8]. In’one of our investigations [6],it was found that the 6,7-dihydroxy-4’-methoxy-isoflavan was fungitoxic to different moulds of the genus Aspergillus. These results led to the conclusion that two hydroxyl
M. WEIDENB~RNER et al.
802
the activity. Also in the present investigation, the isoflavans F,, G,, H, and I, with two or three methoxy groups were almost inactive. However, sativan, a 7hydroxy-2’,4’-dimethoxy-isoflavan was found to be more active than the corresponding 7,2’-dihydroxy-4’methoxy-isoflavan vestitol [16]. In a recent investigation [Ill, it was found that the 6,7-dihydroxy-Y-methyiisoflavan is highly active.
Aa”,
A,Bl
A,A, R' = R' = R' = OH.R) = H B,Bz
R’ =
OMe,
R2 =
OH, R’ =’ H
R’=
EXPERIMENTAL
A3
R’ =
R’
R4 =
OH
M,
R’=
OMc,
R’=
R’=
CI R’=
OMe.
R’ =
H. R’ =
0,
R’ =
R’=
OH,R’=
H.R’=
OMe
tr
R’ =
R3 =
OH.
H. R’ =
OMe
F,
R’=
R’ =
OMe.
R’ =
H. R’ =
OH
(13
R’ =
R’=
OMe.
R’=
H.R’=
Ott
n,
R’ =
OH, R’ =
R’=
R3 =
I3
=
R’
R’ =
=
f,
OH, R3 = R’ =
H Ott
ti, R’ = R4 = OMe
R’ =
OMe.
R’ =
H
and one methoxy group in an isoflavan guarantee a high activity irrespective of the position of the substituents in the molecule, provided one of the rings is disgroups
ubstituted. This is demonstrated by the isoflavans B,, C,, D, and E,, which have two hydroxyl groups and one methoxy group in their molecules and whose ring A is
disubstituted. The importance of derivatising phenolic groups to ethers for increased activity was established by Van Etten [16], Ingham [18], Ingham [19] and Matthews and Van Etten [20]. The presence of an ether function apparently protects the substance against microbial transformations [21]. It was also shown [14, 163 that two hydroxyl groups and one substituted oxygen function other than a methyl-ether group make the isoflavan strongly antifungal. Further methylation could impair
Table
1. Effect of isoflavonoids
on mycelial growth
Isoflaronoids. 13 isoflavonoids were tested in 3 concentrations for their effect on mycelial growth of two soil-borne fungi, R. solani and 8. ro&i. Three compounds belong to the 5,7,4’trihydroxyisoflavone series A, _3, while 5,7-dihydroxy-4’-methoxy-isoflavonoids belong to the series B,-3. The isoflavonoids of each series differ only in the degree of reduction. Besides the isoflavans A, and B, seven other isoflavans C,-I, were also tested (Table 1). In order to study the influence of gradual reduction on the antifungal activity, the isoflavones were catalytically (Pd-C Hz) reduced to isoflavanones and isoflavans. The purity and authenticity of these products were verified by physical methods (NMR and MS). All the substances were synthesized in the Institute of Physiological Chemistry of the University of Bonn. Fungi and culfure. The soil-borne fungi R. .solani and S. rolfsii were originally isolated from Phuseolus ou!yaris and Iris holhdim in German soils near Cologne and kept in the fungus collection of the Institut fur Pflanzenkrankheiten at Bonn. Both fungi are pathogenic to soybeans 1221. The effect of isoflavonoids on my&al growth was investigated in liquid culture. The soln contained 30 g malt extract and 3 g peptone per 1 I distilled H,O. Me&O served as the solvent for the addition of isoflavonoids. The solvent concentration in the soln was maintained at 1.1%. Medium (20ml) including the particular isoflavonoid in the required concentration was transferred to 100 ml Erlenmeyer flasks and inoculated with five small pieces of mycelium (5 mm in diameter). The flasks were incubated at 23-25” on a reciprocal shaker for 7 days. Concentrations of isoJ7avonoid.s. From phytoalexin experiments [l, 231, it is known that concn in the range of 10-5-10-3 M exhibit fungal inhibition. Therefore the concentrations of 0.5, 2.0 and 8.0 x lo-“ M respectively were used in these tests.
of R. solani and S. rolfsii (% difference in wt as compared S. rolfii
R. solani Compound
0.05 mM
A, - 25.0
A, A, B, B, B, C, D, E, F, G, H, I,
-20.6 - 8.7 - 7.9 - 29.9 - 19.7 - 22.9 [( +)indicates
significant
growth
0.2 mM
0.8 mM
-33.5 -16.8 +31.6 -50.1 - 73.4 -40.5 - 79.9 - 10.1 - 16.9 -31.7 - 35.2 -25.7 - 14.6
- 50.0 -79.5 - 79.6 -71.1 - 76.6 - 78.3 - 86.3 - 84.4 - 39.4 -24.2 - 23.5 - 15.7
stimulation,
with the control)
(-)
indicates
significant
0.05 mM -44.5 - 52.4 + 27.7 - 65.4 -26.5 -28.8
f22.8 - 17.5 growth
inhibition].
0.2 mM
0.8 mM
- 64.6 - 58.7
- 66.6 -63.0 -91.6 -90.8 -92.1 - 89.2 -90.8 -94.3 -93.7
‘- 57.0 -92.3 -49.3 -90.9 - 12.5 -23.7 - 36.5 + 20.6 -22.9
- 38.2 - 57.9
Antifungal
activity
Evaluations ofresults. Round filter papers (Schleicher & Schiill No. 595) were weighed after drying for 24 hr at 105”. After filtration of the culture, the residue was dried and weighed as described above. The difference of the weighings gave the dry wt of the fungus. The mean value ofeight repetitions for each concentration and fungus was used for calculation. The data were evaluated by analysis of variance. Probability of single differences was calculated at the 5% level. REFERENCES
I. Kramer, R. P., Hindorf, H., Jha, H. C., Kallage, J. and Zilliken, F. (1984) Phytochemistry 23, 2203. 2. Cruickshank, I. A. M. (,l962) Aust. J. Biol. Sci. 15, 147. 3. Smith, D. A. (1976) Physiol. Plant Pathol. 9, 45. 4. Van Etten, H. D. and Stein, J. I. (1987) Phytopathology 68, 1276. 5. Wyman, J. G. and Van Etten, H. D. (1978) Phytopathology 68, 583. 6. Weidenbiirner, M., Jha, H. C., Hindorf, H., Weltzien, H. C. and Zilliken, F. (1987) Med. Fat. Landbouww. Rijksuniu. Gent. 52, 933. 7. Skipp, R. A. and Bailey, J. A. (1977) Physiol. Plant Pathol. 11, 101. 8. Perrin, D. R. and Cruickshank, I. A. M. (1969) Phytochemistry 8, 971.
of isoflavonoids
803
S. S. and Smith, D. A. (1980) Phytopathol9. Gnanamanickam, ogy 70,894. M., Jha, H. C. and Hindorf, H. (1988) Med. IO. Weidenbiirner, Fat. Landbouww. Rijksuniv. Gent. 53, 1417. M., Hindorf, H., Jha, H. C., Tsotsonos, P. 11. Weidenbiirner, and Egge, H. (1989) Phytochemistry 28, (in press). 12. Adesanya, S. A., O’Neill, M. J. and Roberts, M. F. (1986) Physiol. Plant Pathol. 29, 95. 13. O’Neill, T. M. and Mansfield, J. W. (1982) Trans. Br. Mycol. Sot. 79, 229. 14. Stossel, P. (1985) Physiol. PIant Pathol. 26, 269. 15. Johnson, G., Maag, D. D., Johnson, D. K. and Thomas, R. D. (1976) Physiol. Plant Pathol. 8, 225. 16. Van Etten, H. D. (1976) Phytochemistry 15, 655. 17. Van Etten, H. D. and Pueppke, S. Cl. (1976) in Biochemical Aspects of Plant-Parasite Relationship (Friend, J. and Threlfall, D. R., eds) Ann. Proc. Phytochem. Sot. 13. 18. Ingham, J. L. (1976) Phytochemistry 15, 1489. 19. Ingham, J. L. (1977) Phytochemistry 16, 1279. 20. Matthews, D. E. and Van Etten, H. D. (1983) Arch. Eiochem. Biophys. 224, 494. 21. Sharma, R. D. (1979) Lipids 14, 535. 22. Sinclair, J. B. (1982) Compendium of Soybean Diseases 23. Van Etten, H. D. and Bateman, D. F. (1971) Phytopathology 61. 1363.