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The effect of β-sitosterol on spore germination and germ-tube elongation of Aspergillus niger and Botryodiplodia theobromae
International Journal of Food Microbiology, 8 (1989) 73-78
73
Elsevier J F M 00249
Short C o m m u n i c a t i o n
The effect of fl-sitosterol on spore germination and germ-tube elongation of Aspergillus niger and Botryodiplodia theobromae B a b a t u n d e I. A d e r i y e 1, S a m u e l K. O g u n d a n a l, S a b u r i A. A d e s a n y a a n d M a r g a r e t F. R o b e r t s 3 1 Department of Microbiology, University of Ire, lle-Ife, Nigeria, : Pharmacognosy Department, University of lfe, lie-Ire, Nigeria, and 3 School of Pharmacy, University of London, London, U.K. (Received 4 October 1988; accepted 23 November 1988)
The effect of fl-sitosterol on spore germination and prevention of elongation of germ-tube of Aspergillus niger and Botryodiplodia theobromae was studied. The antifungal activity of the c o m p o u n d showed a percentage inhibition, of the fungal spore germination, of about 40% at a concentration of 50 ~ g / m l while inhibition on the elongation of germ-tubes was as high as 65% at the same concentration. The EDs0 for inhibition of germ-tube elongation in A. niger was about 31 /.tg/ml. The role of the c o m p o u n d in disease resistance is discussed. Key words: fl-Sitosterol; Antifungal activity; Aspergillus niger, Botryodiplodia theobromae; Germination; Germ-tube elongation
Introduction Preformed antifungal compounds occur in the outer tissues of many vegetables such as glycoalkaloids in potato tubers (Katsui et al., 1971; Metlitskii et al., 1971; Ku6, 1972; Tomiyama et al., 1968a; and Varns et al., 1971b) and falcardindiol in carrot roots (Garrod et al., 1978). Antifungal compounds have also been found to accumulate in tissues inflicted or challenged by fungi, e.g. rishitin in potato tubers (Tomiyama et al., 1978a) and 6-methoxymellein in carrot roots (Condon et al., 1963). However, Paxton (1975) noted that both the preformed and induced antifungal compounds play a role in the resistance of plant roots to fungal infection. Numerous type of bioassay tests have utilized to assess the fungitoxic activities of these antimicrobial compounds. These have encompassed effects on germination of spores (McCance and Drysdale, 1975; Tomiyama et al., 1968b), growth of germ tubes (Duczek and Higgins, 1976; Harris and Dennis, 1976 and Higgins, 1978), Correspondence address." Babatunde I. Aderiye, Department of Food Science and Technology, Ondo State Polytechnic, P.M.B. 1019, Owo, Nigeria. 0168-1605/89/$03.50 ~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
74 growth of mycelium across an agar surface (Skipp and Bailey, 1976, 1977) and growth of mycelium in liquid media (Smith, 1976). Selection of an appropriate growth stage of the assay organism is also an important decision. Medicarpin proved toxic to spores and germ-tubes of Colletotrichum phomoides and other fungi (Higgins and Millar, 1968); however, the insensitivity of mycelium rendered this effect more tenuous (Higgins, 1972). This paper reports the effect of fl-sitosterol on some yam rot pathogens and also discusses its possible role, among other natural antifungal compounds from yam peel, in disease resistance in yam tubers (Dioscorea alata L.)
Materials and Methods
Compound fl-sitosterol, one of the compounds extracted from yam peel (D. alata), was found to exhibit antifungal activity on a thin-layer chromatogram bioassay in a previous study (Aderiye, 1987). About 0.43 g of the compound was obtained from 10 kg of yam peel. The compound which had a white crystalline solid appearance was isolated at an R f 0.38 in chloroform : hexane (9 : 1, v/v), purified, characterised and identified as/~-sitosterol (Olugbade, 1979; Aderiye, 1987). The purified extract was always stored under nitrogen gas until needed.
Fungi The fungal pathogens, Aspergillus niger Van Tiegh and Botryodiplodia theobromae Pat. were previously isolated from yam tubers (Ogundana et al., 1970). The organisms were maintained on potato dextrose agar slants (Oxoid) stored at 4 ° C .
Preparation of fungal spore suspension Conidia were harvested in 10 ml distilled water from mature cultures of fungi grown on malt extract agar at 28 ° C for 7 days. The suspension was filtered through two thin layers of sterile non absorbent cotton wool to remove the mycelia fragments and centrifuged. The concentration of the spore suspension was adjusted as required by suspending in a known volume of 2% malt extract broth for bioassay tests (Aderiye, 1984). In the case of B. theobromae, the pycnidia were broken to release the spores (Ekundayo and Haskins, 1969).
Antifungal activity of fl-sitosterol A modification of the method of Coxon et al. (1982) was employed. A known quantity of the spore suspension (4.0 × 105 cells/ml) in 2% malt extract broth was added to 0.5 ml of methanolic solution of/~-sitosterol to give final concentrations of 10, 20, 50 and 100 tLg/ml. The control experiment involved the addition of 0.5 ml of
75
the methanol only to 4.5 ml of the fungal spore suspension. After careful mixing, 0.2 ml of each treatment was pipetted onto sterile glass microscope slides: suspension was retained within a rectangle marked on the slides with molten paraffin wax (Garrod et al., 1978). The prepared slides were incubated in moist Petri dishes at 2 8 ° C for 18 h. The slides were examined every hour for visible germination. Direct counts were made by microscopy to determine the percentage germination. The determination of the mean germ-tube elongation was carried out using the micrometer attached to the ocular lens of the microscope. Here, germinated spores with about 2 /~m long germ-tubes were considered. The spores were allowed to germinate before the addition of the stock solution of the test compound. All the experiments were carried out in triplicate and conducted twice.
Results and Discussion
Effect of ~-sitosterol on fungal spore germination The percentage germination in the control treatments containing methanol was about 79% in Aspergillus niger and 81% in Botryodiplodia theobromae (Table I). The spores of A. niger in the control treatment had grown into branched hyphae after 18 h incubation (Plate la) while a few spores of the same organism in 100 t t g / m l fl-sitosterol have just started germination (Fig. lb). Germination of spores of A. niger appeared to be slightly more affected than spores of B. theobromae (Table I). The EDs0 values were obtained for each species by plotting the percentage inhibition of germination against the log of the concentration of the compound and interpolating. The EDs0 value for inhibition of the germination of spores of A. niger and B. theobromae were 91.2 and 93.3/~g/ml, respectively, a result which confirms the relatively low sensitivity of the spore germination to/3-sitosterol.
Effect of fl-sitosterol on the elongation of germ-tubes The effect of /3-sitosterol on germ-tube elongation was investigated to show whether the compound would stop the already germinated spores from growing
TABLE I Effect of fl-sitosterol on the spore germination of A. niger and B. theobromae Species
EDso
Control a
% Inhibition b 10 / z g / m l
20 t t g / m l
50 t~g/ml
100 ~ g / m l
79 84
14 10
32 25
39 33
51 49
(/~g/ml)
A. niger B. theobromae
91.2 93.3
a Mean % germination in control b Mean % inhibition of germination at concentrations ( # g / m l ) of fl-sitosterol.
76
Fig. 1. (a) Germination of spores of A. niger in the control after 18 h at 28 ° C. (b) Germination of spore: of A. niger in 100 ~ g / m l /3-sitosterol after 18 h at 28 ° C.
77 T A B L E II Effect of fl-sitosterol on the elongation of germ-tubes of A. niger and B. theobromae Species
A. niger B. theobromae
EDs0
Mean germ-tube
% Inhibition ~
(ktg/ml)
elongation in controls (/L m)
10/~g/ml
20 ~ g / m l
50 ~tg/ml
100/zg/ml
30.9 33.1
56 68
10 4
44 42
68 63
83 78
Mean % inhibition of germ-tube elongations at concentrations (/~g/ml) of fl-sitosterol.
further. The results as given in Table II show that the most marked effect was that of the compound on A. niger with percentage inhibition ranging between 10 and 83%. The EDs0 values required to prevent germ-tube elongation of A. niger and B. theobromae were 31 and 33.1 # g / m l , respectively. When compared, germination of spores for both yam rot pathogens was less affected than elongation of germ-tubes by fl-sitosterol. The EDs0 values required to prevent spore germination of the organisms tested were high and similar to those of rishitin when tested against Phytophthora infestans (Harris and Dennis, 1976), Botrytis cinerea and Fusarium avenaceum (Ishizaka et ai., 1969). However, a percentage inhibition of about 83%, at 100 ~ g / m l , of the elongation of germ-tubes of A. niger was obtained. The EDso value required to prevent germ-tube elongation of the yam pathogens were, far lower than those obtained for rishitin (Lyon and Bayliss, 1975). In assessing the importance of chemical inhibitors in disease resistance, the effects on the elongation of germ-tubes are perhaps more relevant, as germination of spores of an infecting fungus would normally have taken place before the penetration of the host tissue (Harris and Dennis, 1976). In this work, fl-sitosterol was found toxic to reproductive fungal structures in liquid media and, in a previous study, to vegetative structures and on thin layer chromatograms (Aderiye, 1987). It therefore seems likely that fl-sitosterol would also be active in intact plants like the peel of the yam, although the supremacy of the compound over the other natural antifungal compounds is yet to be determined.
References Aderiye, B.I. (1984) Effect of ascorbic acid on the incidence growth of fungi associated with Okra (Hibiscus esculentus). Nig. J. Micribiol. 4, 112-116. Aderiye, B.I. (1987) Studies on the natural antifungal compounds from yam tubers (Dioscorea alata L.) Ph.D. thesis, University of Ife, Ile-Ife, Nigeria. Condon, P., Ku6, J. and Draudt, H.N. (1963) Production of 3-methyl-6-methoxy-8-hydroxy-3A-dihydroisocounmarin. Phytopathology 53, 1244-1250. Coxon, D.T., Ogundana, S.K. and Dennis, C. (1982) Antifungal phenanthrenes in yam tubers. Phytochemistry 21, 1389 1392.
78 Duczek, L.J. and Higgms, V.J. (1976) Effect of treatment with the phytoalexins medicarpin and maakiain on fungal growth in vitro and in vivo. Can. J. Bot. 54, 2620-2629. Ekundayo, J.A. and Haskins, R.H. (1969) Pycnidia production of Botryodiplodia theobromae Pat. I. The relation of light to the induction of pycnidia. Can. J. Bot. 47, 1153-1156. Garrod, B., Lewis, B.G. and Coxon, D.T. (1978) Cis-heptadeca-l,9-dienne-4,6-diyne-3,8-diol, an antifungal polyacetylene from carrot root tissue. Physiol. Plant Pathol. 13, 241-246. Harris, J.E. and Dennis, C. (1976) Antifungal activity of post infectional metabolites from potato tubers. Physiol. Plant Pathol., 9, 155 165. Higgins, V.J. (1978) The effect of some pterocarpanoid phytoalexins on germ tube elongation of Stemphylium botryosum Phytopathology 68, 338-345. Higgins, V.J. and Millar, R.L. (1968) Phytoalexin production of alfalafa in response to infection by Colletotrichyn phomoides, Helminthosporium turcicum, Stemphylium loti and S. botryosum. Phytopathology 58, 1377-1383. Ishizaka, N., Tomiyama, K., Katsui, A. and Masamune, T. (1969) Biological activities of rishitin, an antifungal c o m p o u n d isolated from diseased potato tubers and its derivatives. Plant Cell Physiol. 10, 183 192. Katsui, N., Matsunaga, A., lmaizumi, K., Mashamune, T. and Tomiyama, K. (1971) The structure and synthesis of rishitinol, a new sesquiterpense alcohol from diseased potato tubers. Tetrahedron Lett. 2, 83-86. Kug, J. (1972). Phytoalexins. Annu. Rev. Phytopathol. 10, 207-232. Lyon, F.M. and Bayliss, C.E. (1975) The effect of rishitin on Erwinia carotovora var. atroseptica and other bacteria. Physiol. Plant Pathol. 6, 177-186. McCance, D.J. and Drysdale, R.B. (1975) Production of tomatine and rishitin by tomato plants. Physiol. Plant Pathol. 7, 221-230. Metlitskii, L.V., Ozeretskovskaya, O.L., Vul'fson, N.S. and Chalova, L.I. (1971) Chemical nature of lubimin, a new phytoalexin of potatoes. Dokl. Akad. Nauk. S.S.S.R. 200, 1470-1472. Ogundana, S.K., Naqvi, S.H.Z. and Ekundayo, J.A. (1970) Fungi associated with soft rot of yams (Dioscorea spp.) in storage in Nigeria. Trans. Br. Mycol. Soc. 54, 445 451. Olugbade, T.A. (1979) Phytochemical investigation of Cnestis ferruginea D.C. MSc. thesis, University of Ife, lle-Ife, Nigeria. Paxton, J.D. (1975) Biology and Control of Soil-Borne Plant Pathogens. Symposium, September 1973, Minneapolis, MN, Am. Phytopathol. Soc. St. Paul, MN, pp. 185-192. Skipp, R.A. and Bailey, J.A. (1976) The effect of phaseollin on the growth of Colletotrichum linemuthianum is bioassays designed to measure fungitoxicity. Physiol. Plant Pathol., 9, 253-263. Skipp, R.A. and Bailey, J.A. (1977) The fungitoxicity of isoflavonoid phytoalexins measured using different types of bioassay. Physiol. Plant Pathol. 11, 101-112. Smith, D.A. (1976) Some effects of the phytoalexin, Kievitone on the vegetative growth of Aphanomyces euteiches, Rhizoctonia solani and Fusarium solani f. sp. phaseoli. Physiol. Plant Pathol. 9, 45-55. Smith, D.A. (1982) Toxicity of phytoalexins. In: J.A. Bailey and J.W. Mansfield (Eds.), Phytealexins, Blackie and Sons Ltd., Glasgow, pp. 218-252. Tomiyama, K., Ishizaka, N., Sato, N., Masamune, T., and Katusi, N. (1968a) Rishitin, a phytoalexin-like substance, its role in the denfense reaction of potato tubers to infection. In: Biochemical Regulation in Diseased Plants or Injury, Phytopathol. Soc. Japan, Tokyo, pp. 287-292. Tomiyama, K., Sakuma, T., Ishizaka, N., Sato, N., Katusi, N., Takasugi, M. and Mesanune, T. (1968b) A new antifungal substance isolated from resistant potato tuber tissue infected by pathogens. Phytopathology 58, 115-116. Varns, J., Ku6, J. and Williams, E. (1971) Terpenoid accumulatiln as a biochemical response of the potato tuber of Phytophthora infestans. Phytopathology 61, 174-177.
73
Elsevier J F M 00249
Short C o m m u n i c a t i o n
The effect of fl-sitosterol on spore germination and germ-tube elongation of Aspergillus niger and Botryodiplodia theobromae B a b a t u n d e I. A d e r i y e 1, S a m u e l K. O g u n d a n a l, S a b u r i A. A d e s a n y a a n d M a r g a r e t F. R o b e r t s 3 1 Department of Microbiology, University of Ire, lle-Ife, Nigeria, : Pharmacognosy Department, University of lfe, lie-Ire, Nigeria, and 3 School of Pharmacy, University of London, London, U.K. (Received 4 October 1988; accepted 23 November 1988)
The effect of fl-sitosterol on spore germination and prevention of elongation of germ-tube of Aspergillus niger and Botryodiplodia theobromae was studied. The antifungal activity of the c o m p o u n d showed a percentage inhibition, of the fungal spore germination, of about 40% at a concentration of 50 ~ g / m l while inhibition on the elongation of germ-tubes was as high as 65% at the same concentration. The EDs0 for inhibition of germ-tube elongation in A. niger was about 31 /.tg/ml. The role of the c o m p o u n d in disease resistance is discussed. Key words: fl-Sitosterol; Antifungal activity; Aspergillus niger, Botryodiplodia theobromae; Germination; Germ-tube elongation
Introduction Preformed antifungal compounds occur in the outer tissues of many vegetables such as glycoalkaloids in potato tubers (Katsui et al., 1971; Metlitskii et al., 1971; Ku6, 1972; Tomiyama et al., 1968a; and Varns et al., 1971b) and falcardindiol in carrot roots (Garrod et al., 1978). Antifungal compounds have also been found to accumulate in tissues inflicted or challenged by fungi, e.g. rishitin in potato tubers (Tomiyama et al., 1978a) and 6-methoxymellein in carrot roots (Condon et al., 1963). However, Paxton (1975) noted that both the preformed and induced antifungal compounds play a role in the resistance of plant roots to fungal infection. Numerous type of bioassay tests have utilized to assess the fungitoxic activities of these antimicrobial compounds. These have encompassed effects on germination of spores (McCance and Drysdale, 1975; Tomiyama et al., 1968b), growth of germ tubes (Duczek and Higgins, 1976; Harris and Dennis, 1976 and Higgins, 1978), Correspondence address." Babatunde I. Aderiye, Department of Food Science and Technology, Ondo State Polytechnic, P.M.B. 1019, Owo, Nigeria. 0168-1605/89/$03.50 ~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
74 growth of mycelium across an agar surface (Skipp and Bailey, 1976, 1977) and growth of mycelium in liquid media (Smith, 1976). Selection of an appropriate growth stage of the assay organism is also an important decision. Medicarpin proved toxic to spores and germ-tubes of Colletotrichum phomoides and other fungi (Higgins and Millar, 1968); however, the insensitivity of mycelium rendered this effect more tenuous (Higgins, 1972). This paper reports the effect of fl-sitosterol on some yam rot pathogens and also discusses its possible role, among other natural antifungal compounds from yam peel, in disease resistance in yam tubers (Dioscorea alata L.)
Materials and Methods
Compound fl-sitosterol, one of the compounds extracted from yam peel (D. alata), was found to exhibit antifungal activity on a thin-layer chromatogram bioassay in a previous study (Aderiye, 1987). About 0.43 g of the compound was obtained from 10 kg of yam peel. The compound which had a white crystalline solid appearance was isolated at an R f 0.38 in chloroform : hexane (9 : 1, v/v), purified, characterised and identified as/~-sitosterol (Olugbade, 1979; Aderiye, 1987). The purified extract was always stored under nitrogen gas until needed.
Fungi The fungal pathogens, Aspergillus niger Van Tiegh and Botryodiplodia theobromae Pat. were previously isolated from yam tubers (Ogundana et al., 1970). The organisms were maintained on potato dextrose agar slants (Oxoid) stored at 4 ° C .
Preparation of fungal spore suspension Conidia were harvested in 10 ml distilled water from mature cultures of fungi grown on malt extract agar at 28 ° C for 7 days. The suspension was filtered through two thin layers of sterile non absorbent cotton wool to remove the mycelia fragments and centrifuged. The concentration of the spore suspension was adjusted as required by suspending in a known volume of 2% malt extract broth for bioassay tests (Aderiye, 1984). In the case of B. theobromae, the pycnidia were broken to release the spores (Ekundayo and Haskins, 1969).
Antifungal activity of fl-sitosterol A modification of the method of Coxon et al. (1982) was employed. A known quantity of the spore suspension (4.0 × 105 cells/ml) in 2% malt extract broth was added to 0.5 ml of methanolic solution of/~-sitosterol to give final concentrations of 10, 20, 50 and 100 tLg/ml. The control experiment involved the addition of 0.5 ml of
75
the methanol only to 4.5 ml of the fungal spore suspension. After careful mixing, 0.2 ml of each treatment was pipetted onto sterile glass microscope slides: suspension was retained within a rectangle marked on the slides with molten paraffin wax (Garrod et al., 1978). The prepared slides were incubated in moist Petri dishes at 2 8 ° C for 18 h. The slides were examined every hour for visible germination. Direct counts were made by microscopy to determine the percentage germination. The determination of the mean germ-tube elongation was carried out using the micrometer attached to the ocular lens of the microscope. Here, germinated spores with about 2 /~m long germ-tubes were considered. The spores were allowed to germinate before the addition of the stock solution of the test compound. All the experiments were carried out in triplicate and conducted twice.
Results and Discussion
Effect of ~-sitosterol on fungal spore germination The percentage germination in the control treatments containing methanol was about 79% in Aspergillus niger and 81% in Botryodiplodia theobromae (Table I). The spores of A. niger in the control treatment had grown into branched hyphae after 18 h incubation (Plate la) while a few spores of the same organism in 100 t t g / m l fl-sitosterol have just started germination (Fig. lb). Germination of spores of A. niger appeared to be slightly more affected than spores of B. theobromae (Table I). The EDs0 values were obtained for each species by plotting the percentage inhibition of germination against the log of the concentration of the compound and interpolating. The EDs0 value for inhibition of the germination of spores of A. niger and B. theobromae were 91.2 and 93.3/~g/ml, respectively, a result which confirms the relatively low sensitivity of the spore germination to/3-sitosterol.
Effect of fl-sitosterol on the elongation of germ-tubes The effect of /3-sitosterol on germ-tube elongation was investigated to show whether the compound would stop the already germinated spores from growing
TABLE I Effect of fl-sitosterol on the spore germination of A. niger and B. theobromae Species
EDso
Control a
% Inhibition b 10 / z g / m l
20 t t g / m l
50 t~g/ml
100 ~ g / m l
79 84
14 10
32 25
39 33
51 49
(/~g/ml)
A. niger B. theobromae
91.2 93.3
a Mean % germination in control b Mean % inhibition of germination at concentrations ( # g / m l ) of fl-sitosterol.
76
Fig. 1. (a) Germination of spores of A. niger in the control after 18 h at 28 ° C. (b) Germination of spore: of A. niger in 100 ~ g / m l /3-sitosterol after 18 h at 28 ° C.
77 T A B L E II Effect of fl-sitosterol on the elongation of germ-tubes of A. niger and B. theobromae Species
A. niger B. theobromae
EDs0
Mean germ-tube
% Inhibition ~
(ktg/ml)
elongation in controls (/L m)
10/~g/ml
20 ~ g / m l
50 ~tg/ml
100/zg/ml
30.9 33.1
56 68
10 4
44 42
68 63
83 78
Mean % inhibition of germ-tube elongations at concentrations (/~g/ml) of fl-sitosterol.
further. The results as given in Table II show that the most marked effect was that of the compound on A. niger with percentage inhibition ranging between 10 and 83%. The EDs0 values required to prevent germ-tube elongation of A. niger and B. theobromae were 31 and 33.1 # g / m l , respectively. When compared, germination of spores for both yam rot pathogens was less affected than elongation of germ-tubes by fl-sitosterol. The EDs0 values required to prevent spore germination of the organisms tested were high and similar to those of rishitin when tested against Phytophthora infestans (Harris and Dennis, 1976), Botrytis cinerea and Fusarium avenaceum (Ishizaka et ai., 1969). However, a percentage inhibition of about 83%, at 100 ~ g / m l , of the elongation of germ-tubes of A. niger was obtained. The EDso value required to prevent germ-tube elongation of the yam pathogens were, far lower than those obtained for rishitin (Lyon and Bayliss, 1975). In assessing the importance of chemical inhibitors in disease resistance, the effects on the elongation of germ-tubes are perhaps more relevant, as germination of spores of an infecting fungus would normally have taken place before the penetration of the host tissue (Harris and Dennis, 1976). In this work, fl-sitosterol was found toxic to reproductive fungal structures in liquid media and, in a previous study, to vegetative structures and on thin layer chromatograms (Aderiye, 1987). It therefore seems likely that fl-sitosterol would also be active in intact plants like the peel of the yam, although the supremacy of the compound over the other natural antifungal compounds is yet to be determined.
References Aderiye, B.I. (1984) Effect of ascorbic acid on the incidence growth of fungi associated with Okra (Hibiscus esculentus). Nig. J. Micribiol. 4, 112-116. Aderiye, B.I. (1987) Studies on the natural antifungal compounds from yam tubers (Dioscorea alata L.) Ph.D. thesis, University of Ife, Ile-Ife, Nigeria. Condon, P., Ku6, J. and Draudt, H.N. (1963) Production of 3-methyl-6-methoxy-8-hydroxy-3A-dihydroisocounmarin. Phytopathology 53, 1244-1250. Coxon, D.T., Ogundana, S.K. and Dennis, C. (1982) Antifungal phenanthrenes in yam tubers. Phytochemistry 21, 1389 1392.
78 Duczek, L.J. and Higgms, V.J. (1976) Effect of treatment with the phytoalexins medicarpin and maakiain on fungal growth in vitro and in vivo. Can. J. Bot. 54, 2620-2629. Ekundayo, J.A. and Haskins, R.H. (1969) Pycnidia production of Botryodiplodia theobromae Pat. I. The relation of light to the induction of pycnidia. Can. J. Bot. 47, 1153-1156. Garrod, B., Lewis, B.G. and Coxon, D.T. (1978) Cis-heptadeca-l,9-dienne-4,6-diyne-3,8-diol, an antifungal polyacetylene from carrot root tissue. Physiol. Plant Pathol. 13, 241-246. Harris, J.E. and Dennis, C. (1976) Antifungal activity of post infectional metabolites from potato tubers. Physiol. Plant Pathol., 9, 155 165. Higgins, V.J. (1978) The effect of some pterocarpanoid phytoalexins on germ tube elongation of Stemphylium botryosum Phytopathology 68, 338-345. Higgins, V.J. and Millar, R.L. (1968) Phytoalexin production of alfalafa in response to infection by Colletotrichyn phomoides, Helminthosporium turcicum, Stemphylium loti and S. botryosum. Phytopathology 58, 1377-1383. Ishizaka, N., Tomiyama, K., Katsui, A. and Masamune, T. (1969) Biological activities of rishitin, an antifungal c o m p o u n d isolated from diseased potato tubers and its derivatives. Plant Cell Physiol. 10, 183 192. Katsui, N., Matsunaga, A., lmaizumi, K., Mashamune, T. and Tomiyama, K. (1971) The structure and synthesis of rishitinol, a new sesquiterpense alcohol from diseased potato tubers. Tetrahedron Lett. 2, 83-86. Kug, J. (1972). Phytoalexins. Annu. Rev. Phytopathol. 10, 207-232. Lyon, F.M. and Bayliss, C.E. (1975) The effect of rishitin on Erwinia carotovora var. atroseptica and other bacteria. Physiol. Plant Pathol. 6, 177-186. McCance, D.J. and Drysdale, R.B. (1975) Production of tomatine and rishitin by tomato plants. Physiol. Plant Pathol. 7, 221-230. Metlitskii, L.V., Ozeretskovskaya, O.L., Vul'fson, N.S. and Chalova, L.I. (1971) Chemical nature of lubimin, a new phytoalexin of potatoes. Dokl. Akad. Nauk. S.S.S.R. 200, 1470-1472. Ogundana, S.K., Naqvi, S.H.Z. and Ekundayo, J.A. (1970) Fungi associated with soft rot of yams (Dioscorea spp.) in storage in Nigeria. Trans. Br. Mycol. Soc. 54, 445 451. Olugbade, T.A. (1979) Phytochemical investigation of Cnestis ferruginea D.C. MSc. thesis, University of Ife, lle-Ife, Nigeria. Paxton, J.D. (1975) Biology and Control of Soil-Borne Plant Pathogens. Symposium, September 1973, Minneapolis, MN, Am. Phytopathol. Soc. St. Paul, MN, pp. 185-192. Skipp, R.A. and Bailey, J.A. (1976) The effect of phaseollin on the growth of Colletotrichum linemuthianum is bioassays designed to measure fungitoxicity. Physiol. Plant Pathol., 9, 253-263. Skipp, R.A. and Bailey, J.A. (1977) The fungitoxicity of isoflavonoid phytoalexins measured using different types of bioassay. Physiol. Plant Pathol. 11, 101-112. Smith, D.A. (1976) Some effects of the phytoalexin, Kievitone on the vegetative growth of Aphanomyces euteiches, Rhizoctonia solani and Fusarium solani f. sp. phaseoli. Physiol. Plant Pathol. 9, 45-55. Smith, D.A. (1982) Toxicity of phytoalexins. In: J.A. Bailey and J.W. Mansfield (Eds.), Phytealexins, Blackie and Sons Ltd., Glasgow, pp. 218-252. Tomiyama, K., Ishizaka, N., Sato, N., Masamune, T., and Katusi, N. (1968a) Rishitin, a phytoalexin-like substance, its role in the denfense reaction of potato tubers to infection. In: Biochemical Regulation in Diseased Plants or Injury, Phytopathol. Soc. Japan, Tokyo, pp. 287-292. Tomiyama, K., Sakuma, T., Ishizaka, N., Sato, N., Katusi, N., Takasugi, M. and Mesanune, T. (1968b) A new antifungal substance isolated from resistant potato tuber tissue infected by pathogens. Phytopathology 58, 115-116. Varns, J., Ku6, J. and Williams, E. (1971) Terpenoid accumulatiln as a biochemical response of the potato tuber of Phytophthora infestans. Phytopathology 61, 174-177.