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Biochemical changes and phytoalexin accumulation in Phaseolus vulgaris following cellular browning caused by tobacco necrosis virus
Physiological Plant Pathology (1973)
3, 171-177
Biochemical changes and phytoalexin accumulation in Phaseolus vulgaris following cellular browning caused by tobacco necrosis virus J. A.
BAILEY
and R. S.
A.R.C. Plant Growth Wye College (Accepted
Substance
(University
for publication
BURDEN and Systemic Fungicide
Unit,
of Ikwzdon), Wye, Ashford, Kent, U.K. October
1972)
Etiolated bean hypocotyls underwent necrosis and cellular browning following infection by tobacco necrosis virus. This was accompanied by the production of many phenolic compounds, some of which were shown to be highly antifungal in assays of fungal growth on thin-layer plates. Four compounds, phaseollin, phaseollidm, phaseolliisoflavan and kievitone, were isolated and identified. Their miniium lethal doses towards spore germination of Colletotrichum lindemuthianum were 2, 2, 2 and 20 w/ml respectively. In addition, following their isolation in high yield from virus-infected tissue, they were also demonstrated in tissue infected with C. lindemuthianum. They have thus been referred to as phytoalexins. The use of virus-infected tissue as a source of new phytoalexins, the r&le of these compounds in disease resistance and their possible importance in explaining virus-induced resistance to fungal pathogens are discussed.
INTRODUCTION
The purpose of this paper is twofold: firstly, to illustrate the many biochemical changes which occurred in bean tissues (Phase&s vulgaris, L.) which had undergone necrosis and browning in response to infection by tobacco necrosis virus (TNV); and secondly, to report the isolation and properties of three new antifungal compounds (phytoalexins) from both virus-infected tissue and tissue infected with Colletotrichum lindemuthianum (Sacc. & Magn.) Bri. and Cav. Earlier papers have described the close association of phaseollin accumulation and cellular browning in bean caused by C. lindemuthianum [Z], Pseudomonas phaseolicola [14], Uromyces appendiculatus and tobacco necrosis virus [3]. During these studies it was observed from chromatograms of tissue extracts that many other compounds appeared to form at the same time as phaseollin. Other workers [II, 121 have made similar observations and the presence of a second phytoalexin, in addition to phaseollin, has been indicated in beans infected with Rhisoctonia solani [IO]. In the present study, the use of TNV-infected etiolated hypocotyls has permitted the extraction and isolation of large amounts of several new metabolites which have fungitoxic properties. MATERIALS
AND
METHODS
The strains of tobacco necrosis virus and C. lindemuthianum, the cultivar of P. vulgaris, Kievitsboon Koekoek, the preparation of etiolated hypocotyls infected with TNV, and of light-grown hypocotyls infected with C. lindemuthianum, and the extraction of I2
172
J. A. Bailey
and
R. S. Burden
tissue with ethanol have been described previously [Z, 31. Complete partition of phenolic compounds into di-ethyl ether was achieved after adjusting the pH of the aqueous phase to 3.0. Chromatography was carried out on thin layers of silica containing a fluorescent indicator (Merck F254). The solvent systems used were ethanol and chloroform (3 : 100) (solvent A), toluene, ethyl formate and formic acid (7 : 2 : 1) (solvent B), h exane and acetone (3 : 1) (solvent C) and hexane and ethyl acetate (3 : 1) (solvent D). Phenolic compounds were demonstrated by spraying the developed plates with diazotised nitroaniline (DNA) [lg. Gibbs’ reagent [8] was used to differentiate compounds with an unsubstituted CH para to a phenolic hydroxy group. Assays of fungitoxicity were carried out in two ways. In the first, developed silica plates were dried in air and sprayed with a suspension of spores of Cladosporium cucumerinum (Eli. & Arth.) in Czapek Dox solution (Oxoid CM95). The plates were then incubated under high humidity in the dark at 25 “C for between 3 and 6 days. In the absence of inhibitors, the fungus grew extensively on the silica and dark hyphae and spores were clearly visible. Inhibitory zones were revealed as white areas devoid of mycelium. The second assay involved scraping selected bands of silica from the plates, eluting the silica with ethanol and assessing the activity of eluates against germination of fungal spores in 1% malt extract [5]. This latter assay was also used to measure the antifungal activity of pure compounds. RESULTS Demonstration of metabolic changes andformation etiolated bean tissue following infection by lXV
of antifunsal
compounds in
Uninoculated etiolated hypocotyls and hypocotyls which showed extensive cellular browning following inoculation with TNV 5 days earlier were extracted in parallel. Aliquots of each extract (0.1 g fresh tissue for observations under U.V. light and for spraying with DNA; 0.5 g fresh tissue for bioassays) were loaded on to 3-cm wide origins on silica plates, which were then developed in solvent A. Inspection of developed plates under light of wavelength 254 nm revealed numerous dark (light absorbing) bands, particularly in extracts of diseased tissue [Plate 1 (a)]. When the same plates were sprayed with DNA numerous yellow, orange and brown bands appeared immediately. Again these were extremely abundant in the extracts of virus-infected tissue [Plate 1 (b)]. Only two coloured bands, at the origin and at R, 0.10 to 0.15 were apparent in extracts of healthy tissue. When similar plates were examined for antifungal activity, five inhibitory bands were demonstrated in extracts from brown tissue but none from uninoculated tissue (Plate 2). When zones corresponding to these bands of inhibition were scraped from similarly developed chromatograms and eluted with ethanol, the eluates also prevented spore germination of C. cucumerinum. Eluates from plates containing extracts of uninoculated tissue or from apparently non-inhibitory areas on plates containing extracts of virus-infected tissue failed to prevent germination of these spores. The antifungal bands marked 1, 2, 3 and 4 on Plate 2 were subjected to intensive investigation. As a result four antifungal compounds, referred to as 1, 2, 3 and 4 respectively, were isolated. Compound 1 was readily identified, using methods described previously [Z], as phaseollin. Compounds 2 and 3 were separated and
Changes
in P. vulgaris
following
cellular
173
browning
purified by successive chromatography in solvents A and 19. Compound 4 was purified by successive chromatography in solvents A, C and B and after the third chromatographic separation, a single band was apparent at R, 0.22 as a light absorbing band at 254 nm which appeared orange when sprayed with DNA.
OH CH3 CH3 h CH,
CH,
Phaseollidin
Phaseollin
HO
CH3 OH
CH3 Kievitone
Phaseollinisoflavan FIG.
1.
Structures
of antifungal
compounds
in extracts
from
TNV-infected
compounds from
virus-infected
TABLE
Chmical
Chemical
and physical
and physical properties
Ultraviolet absorption spectra I max &OH (log E) A maix KOH Spray reactions DNA [15] Gibbs’ reagent Rf values Solvent A Solvent B Solvent C Solvent D
[8]
proPerties
of antzjhgal
Compound Phaseollin
(4.04) ; 286sh (3.98) 315 (3.42) 253;281;290
1
hypocotyls.
1
Compound Phaseollidin
280
bean
2
bean hypocotyls
Compound Phaseollmisoflavan
Compound Kievitone
281 (3.76) ; 287 (3.80)
280
290
290;
Yellow Pale yellow
Orange Pale yellow
Orange Blue
Orange Purple
0.50 0.56 0.27 0.29
0.37 0.45 0.16 0.17
0.27 0.40 0.17 0.17
0.05 0.22 0.05 0.00
;
(4.01) ; 310sh (3.27)
3
341
293
(4.22) ; 330sh (3.58)
337
4
174
J. A. Bailey
and
R. S. Burden
Using these methods, phaseollin was obtained in crystalline form from aqueous ethanol, while phaseollidin, phaseollinisoflavan and kievitone, which were each chromatographically homogenous in the four solvents listed above, were isolated as syrups. The antifungal activity of these purified compounds was confirmed using both assays, and their properties (Table 1) and structures (Fig. 1) [4] were established. The values for the extinction coefficients (log E at A,,,) of both phaseollin (4.04) and phaseollidin (3.76) obtained in this work (Table 1) are slightly different from those reported previously (3.97 and 3.95, respectively) [G, 91. In our experiments, measurements using phaseollin were carried out with crystalline material, whilst those using phaseollidin were obtained with chromatographically pure samples. Similar results were obtained on at least three occasions. The concentration of these compounds in 15 g of virus-infected hypocotyls was determined spectrophotometrically after obtaining each compound in pure form. The results are shown in Table 2. Concentration
of antifungal
TABLE 2 compounds in browned
O.D.
Compound 1 (phaseollin) Compound 2 ( phaseollidin) Compound 3 (phaseollinisoflavan) Compound 4 (kievitone)
1.38 in 200 ml
Fungal
speciesC
A. brassicicola A. niger B. cinerea C. cucumerinum Colletotrichum lagenarium C. lindemuthianum Glomerella cingulata Septoria nodorum Uromyces fabae
Compound 1 Phaseollin M.L.D.a E.D.50b
with
540
20 ml
66
1.18 in
20 ml
50
0.76 in
20 ml
22
cornpour&
TM’
hypocoW’
0.96 in
TABLE
of antifungal
infected
Concentration at hmax pg/g
a Each compound was obtained from 15-g hypocotyls extinction coefficient. The weight of each hypocotyl
Fungitoxicity
hypocotyls
and the weight calculated from was between 0.5 and 1.0 g.
3 from
virus-infected
Compound 2 Phaseollidin M.L.D.‘J E.D.50b
hypoco&ls
Compound 3 Phaseollinisoflavan M.L.D.a E.D.50b
Compound 4 Kievitone M.L.D.a E.D.50b
> 400 > 400 > 400 50
4.0 100 40 4
> 400 > 400 200 50
40 > 400 20 20
400 > 400 >400 20
20 100 100 4
400 > 400 200 100
10 4 >4Qo 100 10
<2 <2 200 50 >2
4 2 > 400 200 10
<2 <2 40 100 12
10 4 > 400 100 10
4 42 40 20 <2
20 20 200 200 10
(w/ml)
which
completely
growth
by 50%.
a Minimum lethal dose : minimum concentration growth. b Concentration (pg/ml) which reduced germ-tube c See text for full names of these fungi.
the
prevented
40 100 20 20 <2 4 40 100 2
PLATE 1. Occurrence of new compounds in extracts of bean hypocotyls infected with TNV. Extracts of 0.1 g tissue were subjected to t.1.c. on silica in ethanol and chloroform (3 : 100): (a), dried plate photographed under light of wavelength 254 nm; (b), dried plate photographed after spraying with DNA: br-brown, o--orange, y-yellow; (i), uninoculated tissue showing no visible browning; (ii), tissue inoculated with TNV and showing extensive cellular browning. The numbers refer to areas of inhibition on Plate 2.
Changes
in P.
vulgwk following
cellular
175
browning
Similar investigations showed that these compounds were also present in browned hypocotyl tissue following inoculation with either a compatible or an incompatible race of C. lindemuthianum. Fungitoxicity
of antifungal
compounds from
virus-infected
hypocotyls
The effect of the four antifungal compounds on the germination of spores and subsequent germ-tube growth of several fungi is shown in Table 3. The figures represent results which were obtained by comparing, individually, the activity of compounds 2, 3 and 4 with that of phaseollin (Compound 1) in at least two experiments. DISCUSSION
Many compounds which were not detected in uninoculated tissue were found to accumulate in bean hypocotyls which underwent cellular browning following inoculation with tobacco necrosis virus. Several of these compounds were shown to be fungitoxic, both in a direct assay of growth on silica thin-layer plates and also when eluted from the silica and assayed in a spore germination test. Four highly antifungal compounds were isolated and characterized. They all exhibited similar activity against the various fungi used. In contrast to results which indicated that mycelial growth of fungi pathogenic to bean was insensitive to phaseollin [6], the present results revealed that germination of spores of C. lindemuthianum, the only pathogen of bean tested, was completely inhibited by low concentrations of each compound. Several fungi non-pathogenic to bean, e.g. Alternaria tenuis, Aspergillus niger and Botrytis cinerea were unaffected by very much higher levels. The three new antifungal compounds were, like phaseollin [Z], also obtained from tissue which had become brown following inoculation with either a compatible or an incompatible race of C. lindemuthianum and may thus be referred to as phytoalexins. However, evaluation of the role of these compounds in resistance to C. lindemuthianum depends upon knowledge of the amounts present in infected cells at significant stages in the process of infection, as discussed earlier for phaseollin [Z]. Notwithstanding, the presence of these compounds in infected tissue and the extreme sensitivity of C. lindemuthianum suggests that they may augment the action of phaseollin in restricting growth of C. lindemuthianum during a hypersensitive reaction. Assigning trivial names to new natural compounds is difficult when derivations from generic or specific names are not possible. The compounds isolated from inhibitory bands 2 and 3 are structurally related to phaseollin and names can be derived from this source. During the preparation of this paper Perrin et al. [9] reported the structure of phaseollidin, an antifungal compound from beans infected with Monilinia-fructicola. This structure is identical to that of compound 2, as shown in Fig. 1. A comparison of U.V. spectra and mass spectra indicated that they are the same compound [4j. For compound 3, which is not a pterocarpan, the trivial name phaseollinisoflavan is suggested. However, compound 4 is less closely related to these compounds and hence a new name is required. Because it is an isoflavanone and was isolated at Wye from the cultivar of bean Kievitsboon Koekoek, the name kievitone is proposed. Exchange of samples revealed that the same compound (Smith & Bateman, personal communication) has been isolated as a component of substance II [lo] from bean infected with Rhizoctonia solani [13].
J. A. Bailey
176
and
R. S. Burden
The production of antifungal compounds in response to virus infection is a new phenomenon and it must be stated that there is, as yet, no evidence that these or similar compounds play any part in resistance of plants to viruses. However, there are reports that infections by viruses, which result in a necrotic reaction, may protect plants from subsequent fungal attack. For example, tobacco leaves, which had reacted to infection by tobacco mosaic virus or tobacco necrosis virus to produce local necrotic lesions, showed a degree of resistance to subsequent infection by the fungus Thielaviopsis basicola which was absent in leaves which had not received prior treatment with the virus [7]. Similarly, infection of bean (P. vulgaris) leaves by TMV, which again caused local necrotic lesions, also reduced subsequent infection by Uromycesappendiculatus. Observations indicated that this was due to the inhibition of uredospore germination on the leaf surface [16]. Although it was suggested [16] that such effects could be explained in terms of the activity of phytoalexins, it was not then known that viruses could induce the formation of such compounds. The present results and also our unpublished data, which shows that pea and cowpea respond in a similar way, suggest that such protective phenomena may be due to the activity of antifungal compounds produced in necrotic tissue in response to virus infection. However, further experiments are needed to demonstrate the production of such compounds at effective concentrations in protected leaves. Finally, the production of phaseollin and three new phytoalexins in bean tissue as a result of necrosis and browning following infection by either TNV or C. lindemuthianum, together with the demonstration that brown pigments and non-toxic phenolic compounds also accumulated in virus-infected bean, supports the view [I] that the accumulation of phytoalexins in response to infection is part of a general metabolic change. Such changes could be a consequence of cellular activity which accompanies or follows cell death; this in turn being the result of the action of the infecting agent. We wish to thank Dr G. A. Carter and Mrs P. M. Rogers for assistance with bioassays and Dr B. J. Deverall and Professor R. L. Wain, F.R.S., for their helpful advice. REFERENCES 1.
2.
3.
4. 5.
6.
BAILEY, J. A. (1973).
Phaseollin accumulation in Pha-seolw vulgar& following infection by fungi, bacteria and a virus. In FungaC Pathogenicity and the Plant’s Response (R. J. W. Byrde and C. V. Cutting, eds). Academic Press (in press). BAILEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolm vulgaris) and physiological races of Colletotrichum Cindemuthianum. Physiological Plant Pathology 1, 435-449. BAILEY, J. A. & INGHAM, J. L. (1971). Phaseollin accumulation in bean (Phaseolus uulgmis) in response to infection by tobacco necrosis virus and the rust Uromyces apgendiculatus. Physiological Plant Pathology 1, 451456. BURDEN, R. S., BAILEY, J. A. & DAWSON, G. W. (1972). Structures of three new isoflavanoids from Phaseolus vulgaris infected with tobacco necrosis virus. Tetrahedron Letters, 4175-4178. CARTER, G. A., SUMMERS,L. A. & WAIN, R. L. (1972). Investigations on fungicides. XIV. Fungitoxicity of N-(2,2,2-trichloro-I-methoxyethyl) formamide and related compounds. Annals of Applied BioCoD 70, 233-243. CRUICKSHANK,I. A. M. & PERRIN, D. R. (1971). Studies on phytoalexins. XI. The induction, antimicrobial spectrum and chemical assay of phaseollin. Phytofiathologfsche i$tschrift 70, 209229.
Changes in P. vulgaris following
7.
177
cellular browning
HECHT, E. I. 8-z BATEMAN, D. F. (1964). Non-specific acquired from localized infections by Thielaviopsis ba.sicola or viruses
resistance in tobacco
to pathogens resulting leaves. Phytopatholoo
54, 523-530. 8. KING, F. E., KING, T. J. & MANNING, L. C. (1957). An investigation of the Gibbs’ reaction and its bearing on the constitution of jacareubin. 3ournal of the Chemical Society, 1957, C, 563-566. 9. PERRIN, D. R., WHITTLE, C. P. & BATTERHAM, T. J. (1972). Structure of phaseollidin. T&ahedron Letters, 1673-1676. 10. PIERRE, R. E. & BATEMAN, D. F. (1967). Induction and distribution of phytoalexins in Rhizoctonia-infected bean hypocotyls. PhytopathoZoQ 57, 1154-l 160. 11. RAHE, J. E., Ku& J., CHLIANG, C.-M. & WILLIAMS, E. B. (1969). Correlation of phenol metabolism with histological changes in Phaseolw vulgaris inoculated with fungi. .hfeNetherlands Journal of Plant Pathology 75, 58-7 1. 12. ROMANOWSKI, R. D., Ku&, J. & QUACKENBUSH, F. W. (1962). Biochemical changes in seedrings of bean infected with G&etotrichum lindemuthianum. Fhytopathology 52, 1259-1263. 13. SMITH, D. A., VAN ETTEN, H. D. & BATEMAN. D. F. (19711. Isolation of substance II. an antif&gal co£ from Rhiroctonia solani-infected bek &sue. Phytopathologv 61, 912. ’ 14. STHOLASUTA, P., BAILEY, J. A., SEVERIN, V. & DEVERALL, B. J. (1971). Effect of bacterial inoculation of bean and pea leaves on the accumulation of phaseollin and pisatin. Physiological Plant Pathology 1, 177-184. 15. VAN SXJMERE, C. F., WOLF, G., TEUCHY, H. & KINT, J. (1965). A new thin-layer method for phenolic substances and coumarins. Journal of Chromatography 20, 48-60. 16. WILSON, E. M. (1958). Rust-TMV cross protection and necrotic ring reaction in bean. Phytopathology 48, 228-231.
3, 171-177
Biochemical changes and phytoalexin accumulation in Phaseolus vulgaris following cellular browning caused by tobacco necrosis virus J. A.
BAILEY
and R. S.
A.R.C. Plant Growth Wye College (Accepted
Substance
(University
for publication
BURDEN and Systemic Fungicide
Unit,
of Ikwzdon), Wye, Ashford, Kent, U.K. October
1972)
Etiolated bean hypocotyls underwent necrosis and cellular browning following infection by tobacco necrosis virus. This was accompanied by the production of many phenolic compounds, some of which were shown to be highly antifungal in assays of fungal growth on thin-layer plates. Four compounds, phaseollin, phaseollidm, phaseolliisoflavan and kievitone, were isolated and identified. Their miniium lethal doses towards spore germination of Colletotrichum lindemuthianum were 2, 2, 2 and 20 w/ml respectively. In addition, following their isolation in high yield from virus-infected tissue, they were also demonstrated in tissue infected with C. lindemuthianum. They have thus been referred to as phytoalexins. The use of virus-infected tissue as a source of new phytoalexins, the r&le of these compounds in disease resistance and their possible importance in explaining virus-induced resistance to fungal pathogens are discussed.
INTRODUCTION
The purpose of this paper is twofold: firstly, to illustrate the many biochemical changes which occurred in bean tissues (Phase&s vulgaris, L.) which had undergone necrosis and browning in response to infection by tobacco necrosis virus (TNV); and secondly, to report the isolation and properties of three new antifungal compounds (phytoalexins) from both virus-infected tissue and tissue infected with Colletotrichum lindemuthianum (Sacc. & Magn.) Bri. and Cav. Earlier papers have described the close association of phaseollin accumulation and cellular browning in bean caused by C. lindemuthianum [Z], Pseudomonas phaseolicola [14], Uromyces appendiculatus and tobacco necrosis virus [3]. During these studies it was observed from chromatograms of tissue extracts that many other compounds appeared to form at the same time as phaseollin. Other workers [II, 121 have made similar observations and the presence of a second phytoalexin, in addition to phaseollin, has been indicated in beans infected with Rhisoctonia solani [IO]. In the present study, the use of TNV-infected etiolated hypocotyls has permitted the extraction and isolation of large amounts of several new metabolites which have fungitoxic properties. MATERIALS
AND
METHODS
The strains of tobacco necrosis virus and C. lindemuthianum, the cultivar of P. vulgaris, Kievitsboon Koekoek, the preparation of etiolated hypocotyls infected with TNV, and of light-grown hypocotyls infected with C. lindemuthianum, and the extraction of I2
172
J. A. Bailey
and
R. S. Burden
tissue with ethanol have been described previously [Z, 31. Complete partition of phenolic compounds into di-ethyl ether was achieved after adjusting the pH of the aqueous phase to 3.0. Chromatography was carried out on thin layers of silica containing a fluorescent indicator (Merck F254). The solvent systems used were ethanol and chloroform (3 : 100) (solvent A), toluene, ethyl formate and formic acid (7 : 2 : 1) (solvent B), h exane and acetone (3 : 1) (solvent C) and hexane and ethyl acetate (3 : 1) (solvent D). Phenolic compounds were demonstrated by spraying the developed plates with diazotised nitroaniline (DNA) [lg. Gibbs’ reagent [8] was used to differentiate compounds with an unsubstituted CH para to a phenolic hydroxy group. Assays of fungitoxicity were carried out in two ways. In the first, developed silica plates were dried in air and sprayed with a suspension of spores of Cladosporium cucumerinum (Eli. & Arth.) in Czapek Dox solution (Oxoid CM95). The plates were then incubated under high humidity in the dark at 25 “C for between 3 and 6 days. In the absence of inhibitors, the fungus grew extensively on the silica and dark hyphae and spores were clearly visible. Inhibitory zones were revealed as white areas devoid of mycelium. The second assay involved scraping selected bands of silica from the plates, eluting the silica with ethanol and assessing the activity of eluates against germination of fungal spores in 1% malt extract [5]. This latter assay was also used to measure the antifungal activity of pure compounds. RESULTS Demonstration of metabolic changes andformation etiolated bean tissue following infection by lXV
of antifunsal
compounds in
Uninoculated etiolated hypocotyls and hypocotyls which showed extensive cellular browning following inoculation with TNV 5 days earlier were extracted in parallel. Aliquots of each extract (0.1 g fresh tissue for observations under U.V. light and for spraying with DNA; 0.5 g fresh tissue for bioassays) were loaded on to 3-cm wide origins on silica plates, which were then developed in solvent A. Inspection of developed plates under light of wavelength 254 nm revealed numerous dark (light absorbing) bands, particularly in extracts of diseased tissue [Plate 1 (a)]. When the same plates were sprayed with DNA numerous yellow, orange and brown bands appeared immediately. Again these were extremely abundant in the extracts of virus-infected tissue [Plate 1 (b)]. Only two coloured bands, at the origin and at R, 0.10 to 0.15 were apparent in extracts of healthy tissue. When similar plates were examined for antifungal activity, five inhibitory bands were demonstrated in extracts from brown tissue but none from uninoculated tissue (Plate 2). When zones corresponding to these bands of inhibition were scraped from similarly developed chromatograms and eluted with ethanol, the eluates also prevented spore germination of C. cucumerinum. Eluates from plates containing extracts of uninoculated tissue or from apparently non-inhibitory areas on plates containing extracts of virus-infected tissue failed to prevent germination of these spores. The antifungal bands marked 1, 2, 3 and 4 on Plate 2 were subjected to intensive investigation. As a result four antifungal compounds, referred to as 1, 2, 3 and 4 respectively, were isolated. Compound 1 was readily identified, using methods described previously [Z], as phaseollin. Compounds 2 and 3 were separated and
Changes
in P. vulgaris
following
cellular
173
browning
purified by successive chromatography in solvents A and 19. Compound 4 was purified by successive chromatography in solvents A, C and B and after the third chromatographic separation, a single band was apparent at R, 0.22 as a light absorbing band at 254 nm which appeared orange when sprayed with DNA.
OH CH3 CH3 h CH,
CH,
Phaseollidin
Phaseollin
HO
CH3 OH
CH3 Kievitone
Phaseollinisoflavan FIG.
1.
Structures
of antifungal
compounds
in extracts
from
TNV-infected
compounds from
virus-infected
TABLE
Chmical
Chemical
and physical
and physical properties
Ultraviolet absorption spectra I max &OH (log E) A maix KOH Spray reactions DNA [15] Gibbs’ reagent Rf values Solvent A Solvent B Solvent C Solvent D
[8]
proPerties
of antzjhgal
Compound Phaseollin
(4.04) ; 286sh (3.98) 315 (3.42) 253;281;290
1
hypocotyls.
1
Compound Phaseollidin
280
bean
2
bean hypocotyls
Compound Phaseollmisoflavan
Compound Kievitone
281 (3.76) ; 287 (3.80)
280
290
290;
Yellow Pale yellow
Orange Pale yellow
Orange Blue
Orange Purple
0.50 0.56 0.27 0.29
0.37 0.45 0.16 0.17
0.27 0.40 0.17 0.17
0.05 0.22 0.05 0.00
;
(4.01) ; 310sh (3.27)
3
341
293
(4.22) ; 330sh (3.58)
337
4
174
J. A. Bailey
and
R. S. Burden
Using these methods, phaseollin was obtained in crystalline form from aqueous ethanol, while phaseollidin, phaseollinisoflavan and kievitone, which were each chromatographically homogenous in the four solvents listed above, were isolated as syrups. The antifungal activity of these purified compounds was confirmed using both assays, and their properties (Table 1) and structures (Fig. 1) [4] were established. The values for the extinction coefficients (log E at A,,,) of both phaseollin (4.04) and phaseollidin (3.76) obtained in this work (Table 1) are slightly different from those reported previously (3.97 and 3.95, respectively) [G, 91. In our experiments, measurements using phaseollin were carried out with crystalline material, whilst those using phaseollidin were obtained with chromatographically pure samples. Similar results were obtained on at least three occasions. The concentration of these compounds in 15 g of virus-infected hypocotyls was determined spectrophotometrically after obtaining each compound in pure form. The results are shown in Table 2. Concentration
of antifungal
TABLE 2 compounds in browned
O.D.
Compound 1 (phaseollin) Compound 2 ( phaseollidin) Compound 3 (phaseollinisoflavan) Compound 4 (kievitone)
1.38 in 200 ml
Fungal
speciesC
A. brassicicola A. niger B. cinerea C. cucumerinum Colletotrichum lagenarium C. lindemuthianum Glomerella cingulata Septoria nodorum Uromyces fabae
Compound 1 Phaseollin M.L.D.a E.D.50b
with
540
20 ml
66
1.18 in
20 ml
50
0.76 in
20 ml
22
cornpour&
TM’
hypocoW’
0.96 in
TABLE
of antifungal
infected
Concentration at hmax pg/g
a Each compound was obtained from 15-g hypocotyls extinction coefficient. The weight of each hypocotyl
Fungitoxicity
hypocotyls
and the weight calculated from was between 0.5 and 1.0 g.
3 from
virus-infected
Compound 2 Phaseollidin M.L.D.‘J E.D.50b
hypoco&ls
Compound 3 Phaseollinisoflavan M.L.D.a E.D.50b
Compound 4 Kievitone M.L.D.a E.D.50b
> 400 > 400 > 400 50
4.0 100 40 4
> 400 > 400 200 50
40 > 400 20 20
400 > 400 >400 20
20 100 100 4
400 > 400 200 100
10 4 >4Qo 100 10
<2 <2 200 50 >2
4 2 > 400 200 10
<2 <2 40 100 12
10 4 > 400 100 10
4 42 40 20 <2
20 20 200 200 10
(w/ml)
which
completely
growth
by 50%.
a Minimum lethal dose : minimum concentration growth. b Concentration (pg/ml) which reduced germ-tube c See text for full names of these fungi.
the
prevented
40 100 20 20 <2 4 40 100 2
PLATE 1. Occurrence of new compounds in extracts of bean hypocotyls infected with TNV. Extracts of 0.1 g tissue were subjected to t.1.c. on silica in ethanol and chloroform (3 : 100): (a), dried plate photographed under light of wavelength 254 nm; (b), dried plate photographed after spraying with DNA: br-brown, o--orange, y-yellow; (i), uninoculated tissue showing no visible browning; (ii), tissue inoculated with TNV and showing extensive cellular browning. The numbers refer to areas of inhibition on Plate 2.
Changes
in P.
vulgwk following
cellular
175
browning
Similar investigations showed that these compounds were also present in browned hypocotyl tissue following inoculation with either a compatible or an incompatible race of C. lindemuthianum. Fungitoxicity
of antifungal
compounds from
virus-infected
hypocotyls
The effect of the four antifungal compounds on the germination of spores and subsequent germ-tube growth of several fungi is shown in Table 3. The figures represent results which were obtained by comparing, individually, the activity of compounds 2, 3 and 4 with that of phaseollin (Compound 1) in at least two experiments. DISCUSSION
Many compounds which were not detected in uninoculated tissue were found to accumulate in bean hypocotyls which underwent cellular browning following inoculation with tobacco necrosis virus. Several of these compounds were shown to be fungitoxic, both in a direct assay of growth on silica thin-layer plates and also when eluted from the silica and assayed in a spore germination test. Four highly antifungal compounds were isolated and characterized. They all exhibited similar activity against the various fungi used. In contrast to results which indicated that mycelial growth of fungi pathogenic to bean was insensitive to phaseollin [6], the present results revealed that germination of spores of C. lindemuthianum, the only pathogen of bean tested, was completely inhibited by low concentrations of each compound. Several fungi non-pathogenic to bean, e.g. Alternaria tenuis, Aspergillus niger and Botrytis cinerea were unaffected by very much higher levels. The three new antifungal compounds were, like phaseollin [Z], also obtained from tissue which had become brown following inoculation with either a compatible or an incompatible race of C. lindemuthianum and may thus be referred to as phytoalexins. However, evaluation of the role of these compounds in resistance to C. lindemuthianum depends upon knowledge of the amounts present in infected cells at significant stages in the process of infection, as discussed earlier for phaseollin [Z]. Notwithstanding, the presence of these compounds in infected tissue and the extreme sensitivity of C. lindemuthianum suggests that they may augment the action of phaseollin in restricting growth of C. lindemuthianum during a hypersensitive reaction. Assigning trivial names to new natural compounds is difficult when derivations from generic or specific names are not possible. The compounds isolated from inhibitory bands 2 and 3 are structurally related to phaseollin and names can be derived from this source. During the preparation of this paper Perrin et al. [9] reported the structure of phaseollidin, an antifungal compound from beans infected with Monilinia-fructicola. This structure is identical to that of compound 2, as shown in Fig. 1. A comparison of U.V. spectra and mass spectra indicated that they are the same compound [4j. For compound 3, which is not a pterocarpan, the trivial name phaseollinisoflavan is suggested. However, compound 4 is less closely related to these compounds and hence a new name is required. Because it is an isoflavanone and was isolated at Wye from the cultivar of bean Kievitsboon Koekoek, the name kievitone is proposed. Exchange of samples revealed that the same compound (Smith & Bateman, personal communication) has been isolated as a component of substance II [lo] from bean infected with Rhizoctonia solani [13].
J. A. Bailey
176
and
R. S. Burden
The production of antifungal compounds in response to virus infection is a new phenomenon and it must be stated that there is, as yet, no evidence that these or similar compounds play any part in resistance of plants to viruses. However, there are reports that infections by viruses, which result in a necrotic reaction, may protect plants from subsequent fungal attack. For example, tobacco leaves, which had reacted to infection by tobacco mosaic virus or tobacco necrosis virus to produce local necrotic lesions, showed a degree of resistance to subsequent infection by the fungus Thielaviopsis basicola which was absent in leaves which had not received prior treatment with the virus [7]. Similarly, infection of bean (P. vulgaris) leaves by TMV, which again caused local necrotic lesions, also reduced subsequent infection by Uromycesappendiculatus. Observations indicated that this was due to the inhibition of uredospore germination on the leaf surface [16]. Although it was suggested [16] that such effects could be explained in terms of the activity of phytoalexins, it was not then known that viruses could induce the formation of such compounds. The present results and also our unpublished data, which shows that pea and cowpea respond in a similar way, suggest that such protective phenomena may be due to the activity of antifungal compounds produced in necrotic tissue in response to virus infection. However, further experiments are needed to demonstrate the production of such compounds at effective concentrations in protected leaves. Finally, the production of phaseollin and three new phytoalexins in bean tissue as a result of necrosis and browning following infection by either TNV or C. lindemuthianum, together with the demonstration that brown pigments and non-toxic phenolic compounds also accumulated in virus-infected bean, supports the view [I] that the accumulation of phytoalexins in response to infection is part of a general metabolic change. Such changes could be a consequence of cellular activity which accompanies or follows cell death; this in turn being the result of the action of the infecting agent. We wish to thank Dr G. A. Carter and Mrs P. M. Rogers for assistance with bioassays and Dr B. J. Deverall and Professor R. L. Wain, F.R.S., for their helpful advice. REFERENCES 1.
2.
3.
4. 5.
6.
BAILEY, J. A. (1973).
Phaseollin accumulation in Pha-seolw vulgar& following infection by fungi, bacteria and a virus. In FungaC Pathogenicity and the Plant’s Response (R. J. W. Byrde and C. V. Cutting, eds). Academic Press (in press). BAILEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolm vulgaris) and physiological races of Colletotrichum Cindemuthianum. Physiological Plant Pathology 1, 435-449. BAILEY, J. A. & INGHAM, J. L. (1971). Phaseollin accumulation in bean (Phaseolus uulgmis) in response to infection by tobacco necrosis virus and the rust Uromyces apgendiculatus. Physiological Plant Pathology 1, 451456. BURDEN, R. S., BAILEY, J. A. & DAWSON, G. W. (1972). Structures of three new isoflavanoids from Phaseolus vulgaris infected with tobacco necrosis virus. Tetrahedron Letters, 4175-4178. CARTER, G. A., SUMMERS,L. A. & WAIN, R. L. (1972). Investigations on fungicides. XIV. Fungitoxicity of N-(2,2,2-trichloro-I-methoxyethyl) formamide and related compounds. Annals of Applied BioCoD 70, 233-243. CRUICKSHANK,I. A. M. & PERRIN, D. R. (1971). Studies on phytoalexins. XI. The induction, antimicrobial spectrum and chemical assay of phaseollin. Phytofiathologfsche i$tschrift 70, 209229.
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HECHT, E. I. 8-z BATEMAN, D. F. (1964). Non-specific acquired from localized infections by Thielaviopsis ba.sicola or viruses
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54, 523-530. 8. KING, F. E., KING, T. J. & MANNING, L. C. (1957). An investigation of the Gibbs’ reaction and its bearing on the constitution of jacareubin. 3ournal of the Chemical Society, 1957, C, 563-566. 9. PERRIN, D. R., WHITTLE, C. P. & BATTERHAM, T. J. (1972). Structure of phaseollidin. T&ahedron Letters, 1673-1676. 10. PIERRE, R. E. & BATEMAN, D. F. (1967). Induction and distribution of phytoalexins in Rhizoctonia-infected bean hypocotyls. PhytopathoZoQ 57, 1154-l 160. 11. RAHE, J. E., Ku& J., CHLIANG, C.-M. & WILLIAMS, E. B. (1969). Correlation of phenol metabolism with histological changes in Phaseolw vulgaris inoculated with fungi. .hfeNetherlands Journal of Plant Pathology 75, 58-7 1. 12. ROMANOWSKI, R. D., Ku&, J. & QUACKENBUSH, F. W. (1962). Biochemical changes in seedrings of bean infected with G&etotrichum lindemuthianum. Fhytopathology 52, 1259-1263. 13. SMITH, D. A., VAN ETTEN, H. D. & BATEMAN. D. F. (19711. Isolation of substance II. an antif&gal co£ from Rhiroctonia solani-infected bek &sue. Phytopathologv 61, 912. ’ 14. STHOLASUTA, P., BAILEY, J. A., SEVERIN, V. & DEVERALL, B. J. (1971). Effect of bacterial inoculation of bean and pea leaves on the accumulation of phaseollin and pisatin. Physiological Plant Pathology 1, 177-184. 15. VAN SXJMERE, C. F., WOLF, G., TEUCHY, H. & KINT, J. (1965). A new thin-layer method for phenolic substances and coumarins. Journal of Chromatography 20, 48-60. 16. WILSON, E. M. (1958). Rust-TMV cross protection and necrotic ring reaction in bean. Phytopathology 48, 228-231.