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Influence of light and excision of organs on accumulation of phytoalexins in virus-infected hypocotyls of bean (Phaseolus vulgaris)
PhJs~iotqicat Plant Patholo~ (1982) 21, 75-84
Influence of light and excision of organs on accumulation of phytoalexins in virus-infected hypocotyls of bean (Phaseohs vulgaris) P.
M.
ROWELL
and J. A. BAILEY
Long Ashton ResearchStation, University of Bristol,
Long Ashton,
Bristol BS18 9AF, U.K.
(Accq!&dforpublication March 1982)
The phytoalexins, phaseollin, phaseollidin, phaseollinisolIavan and kievitone, were isolated from bean hypocotyls bearing TNV-induced necrotic lesions. Phaseollin was the predominant phytoalexin and was localized within the necrotic tissues. Highest yields of phase&n (more than 450 pg g-1 fresh wt) were obtained from dark-grown infected bean seedlings brought into light. The production of each phytoalexin was reduced when cotyledons were removed. Gxnvth and incubation in total darkness completely prevented the production of any phaseollinisoflavan. The total amount of virus produced was not greatly affected by these treatments. It is suggested that the amounts of phytoalexin which accumulate depend on the different biochemical and physiological activities of the living tissues with which adjacent necrotic tissues interact.
INTRODUCTION Phytoalexins accumulate in plants in response to injurious stimuli [Z], including infections by viruses which cause formation of local necrotic lesions. Studies have been reported with virus-infected Phaseolus vulgaris, Glycine max, Pisum sativum, Vigna sinenrti, Canavalia ensiformis and several species of Nicotiana [2, 6, 16, 17, 231. Four phytoalexins, phaseollin, phaseollidin, phaseollinisoflavan and kievitone, were isolated from tobacco necrosis virus (TNV)-infixted bean hypocotyls [3]. This tissue was later used as a source of the phytoaiexins for studies on their metabolism ‘by fungi and plants and for assessing their antimicrobial activity [5]. Yields of phytoalexins from TNV-infected hypocotyls varied considerably, from O-01 to 2 mg g -I tissue. The present investigation was aimed at defining optimum conditions for the accumulation of phytoalexins in bean seedlings. Subsequently the influence of light and the relative contribution of leaves, roots and cotyledons were studied. MATERIALS AND METHODS Virus and plant material Tobalcco necrosis virus was obtained as described previously [3]. However, on this occasion the virus suspension was prepared from living rather than frozen tissue, since the virus titre was found to be much greater in living tissues than in those stored1 at -20 “C. All seedlings were grown in complete darkness from seeds of Phaseoh vulgaris, cv. Prince, sown in seed trays containing sterilized vermiculite which was kept moist 0048-4059/82/040075
+ 10 $03.00/O
@
1982 Academic
Press
Inc.
(London)
Limited
76
P. M. Rowell
and J. A. Bailey
by adding tap water only. Darkness was ensured by placing the trays, immediately after seed sowing, in boxes which totally excluded light in a dark room protected by a light trap. They were incubated at 22&l “C. Inoculation and incubation of bean hypocotyl tissue Hypocotyls (15 to 20 cm long) of 7-day-old seedlings were inoculated by rubbing with foam sponge soaked with a suspension of TNV containing Carborundum; The virus titre was adjusted to give browning throughout the hypocotyls without causing the tissues to wilt or desiccate. Inoculations were carried out under green light and within 5 min the hypocotyls were washed with water. Inoculated seedlings were incubated intact or after excision of various tissues. All cut surfaces were sealed with petroleum jelly. Seedlings lacking roots and excised hypocotyls were placed on their sides in transparent plastic boxes; those retaining their roots remained in the vermiculite. Inoculated tissues were incubated at 22fl “C, either in total darkness (see above) or in a growth room illuminated for periods of 16 h with fluorescent light (8 to 10 klx) followed by 8 h darkness. ” Measurement of phytoalexin concentration Sections (10 cm) cut from the middle of inoculated hypocotyls were used for analysis of phytoalexins; Each sample usually consisted of 7 sections, each weighing approximately 1 g. Phytoalexins were obtained by methods based on those used previously [4]. Tissue was macerated in ethanol and after evaporation the aqueous phase was partitioned with di-ethyl ether. Initial separation of the ether extract was achieved on silica thin layers (Merck 5715, 0.25 mm) using chloroform and ethanol (100 : 4, v/v). This solvent led to purification of phaseollin, phaseollidin and phaseollinisoflavan. Kievitone was purified by subsequent repeated development with chloroform and ethanol (100 : 5). The phytoalexins were eluted from the silica with ethanol and their concentrations measured by ultraviolet spectroscopy. Losses during purification were estimated to be approximately 25% for phaseollin, phaseollidin and phaseollinisoflavan and 35% for kievitone. Diazotized p-nitroaniline (DpNA) was used to detect trace amounts of phytoalexins on chromatograms. The final concentrations of phytoalexins are usually expressed in terms of their weight per hypocotyl. This indicates total yield of phytoalexin and avoids problems associated with loss of tissue weight (dehydration) during later stages of symptom development. Measureementof virus titre Two sections (10 cm) of hypocotyl tissue were macerated in 2 ml of distilled water. The mixture was filtered through muslin and a little Carborundum was added to the filtrate. The filtrate was used to inoculate the 3rd and 4th leaves of two, 8 to lo-weekold plants of Ncotiana tabacum cv. White Burley. The average number of lesions on half of each leaf was recorded 8 days later. RESULTS
Seedlings grown in total darkness for 7 days consisted of a white hypocotyl, 15 to 20 cm long, with small pale yellow leaves contained between the cotyledons. Continued incubation of seedlings in darkness led to extensive elongation of the epicotyl (10 to
Light and accumulation of phytoalexins
77
20 cm) but the leaves remained small and did not expand greatly; there was no green pigmentation. When transferred to light, all the tissues of the seedlings turned green; the epicotyl remained short (less than 5 cm) and the leaves expanded. Excised hypocotyls, i.e. lacking cotyledons, appeared identical when kept in darkness or transferred to light, showing a complete lack of green pigments. Hypocotyls bearing cotyledons always turned green when placed in light. Effect
of light
on accumulation
of phytoalexinr
In darkness, discrete brown lesions were first observed 32 to 37 h after inoculation on both intact seedlings and on excised hypocotyl sections. In light, brown lesions did not develop until 10 to 12 h later. Further incubation led to increased browning, until the entire hypocotyl tissues were discoloured. As in previous investigations [3], the production of phytoalexins was closely associated with symptom development and phaseollin was concentrated within the lesion tissue (Table 1). Only traces of this phytoalexin were detected in tissue immediately surrounding the lesions and none could be found in the unaffected tissue between the lesions. Thin layer chromatograms of these extracts indicated that phaseollidin and kievitonewere distributed similarly. TABLE
Distribution
Tissue
of phaseollin
hypoco@ls of intact seedlings
Tissue Wt (9)
extracted
Necrotic lesions Tissute adjacent to necrotic Remaining tissue Entire hypocotyls
!
in TM’-infected
tissue
1.21 1.61 1.41 1.93
Amount (I.@) 195 t n.d. 96
of phaseollin (f.tg g-r tissue) 174 t n.d. 50
Seven-day-old dark-grown seedlings were inoculated with TNi and incubated in darkness for 3 days, at which time discrete superficial lesions were present. t, Phaseollin was detected on t.1.c. plates with DpNA: amount present was less than 5 ug; nd., phaseollin was not detected with DpNA: limit of detection was less than 0.5 pg.
A, comparison of phytoalexin concentrations in intact seedlings and in excised hypocotyl sections incubated in light or darkness is illustrated in Fig. 1. Phaseollin accumulated more quickly during the initial stages of symptom development in hypocotyls incubated in darkness [Fig. 1 (a) and (c)] than in light [Fig. 1(b) and (d)]. In darkness, the concentrations of all phytoalexins, in both intact seedlings and excised hypocotyl sections, increased to a maximum after 6 to 8 days, when hypocotyls of intact seedlings contained approximately twice as much of each phytoalexin as excised sections. After 8 days slight reductions in phytoalexin concentrations occurred. In light, the accumulation of phaseollidin and kievitone in both intact seedhngs and excised hypocotyls followed a pattern similar to that found in darkness, although kievitone appeared to be produced earlier in excised hypocotyls. The major effect of light was observed with intact seedlings, where the production of phase:ollin continued for more than 8 days, such that after nearly 10 days the yield of phase:ollin exceeded 450 pg per hypocotyl; a concentration which was more than 6 times that obtained from excised hypocotyls incubated in light and, as above, was
P. M. Rowell end J. A. Bailey
78
(d)
6 Perlod
of incubotlon
8
(h 1
FIG. 1. Accumulation of phytoalexins in TNV-infected hypocotyl tissue. Hypocotyls of dark-grown bean seedlings were inoculated with TNV. Intact seedlings or excised hypocotyl sections were incubated in light or darkness. Phytoalexin concentrations in hypocotyl tissue (b) Hypocotyls were measured at intervals. (a) Hypocotyls of seedlings incubated in darkness. of seedlings incubated in light. (c) Excised hypocotyl sections incubated in darkness. (d) Excised hypocotyl sections incubated in light. 0, Phaseollin; A, phaseollidin; X, kicvitonc; 0, phaseollinisoflavan.
Light and accumulation of phytoalexins
79
twice that obtained from intact seedlings incubated in darkness. In contrast, however, excised hypocotyl sections produced 75 jtg phaseollin per hypocotyl when incubated in light and this was less than that found in excised sections maintained in darkness (129 pg per hypocotyl). In all these experiments the maximum concentrations of phaseollidin and kievitone were less than those of phaseollin, varying between 19 and 86 l,tg per hypocotyl. Phaseollinisoflavan was not detected in tissues maintained in complete darkness. Aftler incubation in light this phytoalexin was present in trace amounts in excised hypocotyl sections, and in greater quantities (50 pg per hypocotyl) in entire seedlings, especially when incubated for a long time (9 to 14 days). Con,tribution of cotyledons,leavesand roots to production of phytoalexins The previous results indicate that hypocotyls of intact seedlings produce greater quantities of phaseollin when incubated in light than in darkness and that, in light, inta.ct seedlings produce much more phaseollin than excised hypocotyl sections. The following experiments assessedthe contribution of cotyledons, leaves and/or roots to the accumulation of phytoalexins in bean hypocotyls. Hypocotyls were inoculated with TNV, the appropriate organs were removed and the remaining tissue incubated in 1:ight or darkness for 7 days. No differences in the time of symptom appearance were observed with hypocotyls of intact seedlings and those lacking cotyledons, roots and/or leaves. The results are presented in Tables 2 and 3. As in the earlier experiments (see Fig. l), the amounts of phytoalexin produced in tissues incubated in light or darkness did not differ greatly at the 7 day sampling stage. :Removing both cotyledons caused substantial reductions, to between one-half and one-fifth, in the concentrations of phaseollin and phaseollidin produced in either light or darkness (Table 2). Similar reductions occurred if the leaves were also Eff&
Tissue
of removing
removed
None Two cotyledons All leaves Two cotyledons and all leaves One ~cotyledon Two half cotyledons One and a halfcotyledons Two, threequarter cotyledons One and a threequarter cotyledons
cotyledonous
TABLE 2 tissue on the accumulation seea%&
Phytoalexin Phaseollin Dark Light
of phytoalexins
in hypOcolylE
of
bean
concentration (ug/hypocotyl) Phaseollidin Phaseoilinisoflavan Dark Light Dark Light 73.0*2.0 23.0&3.0 735*0*5
69-O&9.0 30.5*0.5 86*5*7.5
n.d. n.d. n.d.
11.5*0*5 4-F 19.0’
89.555.5 85.5& 14.5 184*0&4.0 208.0*13*0 136.5&l 1.5 1965*26.6
27.055.0 65.5&6.5 46.0 *9-O
36.563.5 76*5+8-5 72.5 ho.5
n.d. n.d. nd.
3.0+ 1.0 10*0*0 ll*o*l~o
135*5+7.5
179.0*22.1
54.0&10.0
67.0&15.0
n-d.
8.0*
1.0
121.542.5
174.5+25.6
41.0+6.0
64.0&
n.d.
7*0*
1.0
9O.Ok5.0
171.5*31*6
35.54
n.d.
7*0*1*0
205.0&8*0 93*5*2-5 207.Ok9.0
259.5 i30.6 141.5*14.5 231.0*29*1
1.5
59-O&6.0
14.0
n.d.. Phaseollinisoflavan not detected with DpNA : limit of detection was less than 0.5 fig. Se&day-old dark-grown seedlings were inoculated with TNV. Cotyledonous &sue was removed, and the seedlingsincubated in light or darkness. Samples of 7 hypoeetyls sections were extracted after 8 days. Values are means and standard errors of 2 experiments, except where indicated.U
80
P. M. Row4 and J. A. Bailey
removed but removal of the leaves alone caused no effect. Phaseollinisoflavan was again not detected in any tissues incubated in darkness. It was present in all tissue incubated in light, but removing cotyledons reduced production of phaseollinisoflavan to one-quarter. Accumulation of phaseollinisoflavan was greatest in seedlings lacking leaves ; an effect which was observed in several other experiments [ZO]. The contributions of different amounts of cotyledonous tissue to the accumulation of phytoalexins are also presented in Table 2. In darkness, removal of increasing portions of cotyledonous tissue caused a corresponding reduction in the concentrations of phaseollin and phaseollidin which formed. This correlation was not observed in seedlings incubated in light; provided a small portion of one cotyledon remained intact, the quantity of phytoalexin produced remained constant, at between 70 to 75% of that found in intact seedlings. Removing roots had very little effect on the amounts of phytoalexin produced (Table 3). TABLE
Effect of removal of roots and other organs on accumlaGon
3
of phaseollin in hypootyls of beansedlings Phase&n concentration Dar~ hyPocoW-l)
Tissue None Roots Roots Roots Roots,
removed
and cotyledons and leaves cotyledons and leaves
Light 193*5* 10-5 206-O& 14.0 73*0*13*0 15&O&26.1 63.5hl5.6
201*5*27-6 192*0*15.0 100*5*15.0 190.5k22.8 85*5&4-5
Seven-day-old dark grown seedlings were inoculated with TNV. Roots, cotyledons and/or leaves were removed, cut surfaces sealed with petroleum jelly and the remaining tissue was incubated in light or darkness. Samples of 7 hypocotyl sections were extracted after 8 days. The values are means and standard errors of 2 separate experiments.
Effect of physiological factors on virus multiplication The number of infective virus particles in hypocotyl tissues inoculated with TNV and incubated in darkness or light is shown in Table 4, which also reports the amounts of phaseollin produced. Intact seedlings maintained constant titres for at least 7 days. Removing cotyledons and/or leaves did not effect the virus titre, but lack of roots, cotyledons and leaves caped a reduction in titre after incubation for 7 days. Differences in phaseollin concentration showed similar trends to those found in earlier experiments. No relationship was apparent between virus titre and phaseollin accumulation.
DISCUSSION Prior to inoculation with TNV, all bean seedlings were grown in complete darkness. This ensured that the hypocotyl tissue was susceptible to virus infection and replication [7, 8, II, 151, but more importantly it allowed an assessment of the influence of light on the subsequent accumulation of phytoalexins.
two
Of
and
40.3hl.8 -
34.8k3.1
Dark
3 Days
h+opl
34.8*5.9 -
26.3 k4.0
Light
incubated
T-U 4 in light or dknkess
20.8-14.1 -
37~3*5.6
leaf
17.0&2.7 36.3k5.7 39.3 54.3 36.8A4.1
34.3h5.5
Dark
14.8k2.8 40.3 f 7.6 35.Q 1.8 33.8k4.0
42.553.3
Light
and/or
leaves were removed, taken for measurement
7 Days
and after removal of roots, cotyledons
were inoculated with TNV. Roots, cotyledons and/or incubated in light or darkness. Samples of tissue were
33.3+2.8 -
35.3s3.8
Number of lesions per half of &otiana tab&m 5 Days Dark Light
tissue of seedlings
Hypocotyls of 7-day-old dark grown seedlings with petroleum jelly and the remaining tissue phaseollm concentrations.
and
cotyledons
TAT-incubakd
leaves
virus from
removed
Two cotyledons Leaves Two cotyledons
IfZlVeS
None Roots,
Tissue
Ti&e
concentration 7 Days
83 84 132 63
163
Light
cut surfaces sealed of virus titre and
87 81 145 77
182
Dark
his bwcovl - ‘1
Phase&in
leaves
82
P. M. Rowell and J. A. Bailey
The influence of host physiology on production of phytoalexins has not received much attention, although greater physiological activity, e.g. as found in younger rather than senescent tissues [I], in tissues incubated at higher temperatures [19], in high oxygen concentrations [9] or in light [18], leads to increased accumulation of phytoalexins. In the present experiments, greatest yields of each phytoalexin were obtained from hypocotyls of intact seedlings incubated in light. High concentrations resulted from applying a virus suspension that caused the entire tissue to become brown but allowed it to remain turgid for a long time. Under these conditions a period of between 7 and 10 days was needed for maximum yields, and slight reductions in the concentrations of phaseollin, phaseollidin and kievitone occurred when seedlings were incubated longer. If the virus suspension was more concentrated, yields of phytoalexin were much reduced due to early wilting and desiccation of the entire hypocotyl [21]. When the contributions of individual factors responsible for the high yields of phytoalexins were studied, it was found that incubation in light with cotyledons remaining intact produced greatest yields. Light also increased accumulation of glyceollins in soybean seedlings infected with Phytophthora megasperma [l&j. In excised bean hypocotyl sections, however, less phytoalexin was produced in light than in darkness. This may be associated with delayed symptom expression such that phytoalexin synthesis occurs when the biochemical reserves of the tisuse have been reduced. The presence of both cotyledons led to greatest accumulation of phytoalexins in the hypocotyls. In darkness removing increasing amounts of cotyledonous tissue produced a progressive decline in the amount of phytoalexin produced. Thus the presence of a quarter of one cotyledon increased phytoalexin accumulation only slightly. In light, however, a quarter of one cotyledon was sufficient to double the yield found in seedlings lacking both cotyledons. The presence or absence of roots did not much affect yield. This contrasts with studies on production of phaseollin in wounded bean seedlings by Rahe & Arnold [20], which showed that removing roots decreased phytoalexin formation. Research with both bean and soybean hypocotyls has also shown that tissue adjacent to the roots produces more phytoalexin than tissue adjacent to the cotyledons [20,24]. In the present study such effects were avoided by excising tissue from the centre of the etiolated hypocotyls. The close association between cell death and accumulation of phytoalexins was discussed previously [2] and was confirmed in the present work. It has been proposed that phytoalexins accumulate in tissues as a consequence of localized cell injury which initiates an interaction between injured and adjacent living cells. This interaction leads to synthesis of phytoalexins in the living tissue from where they diffuse to accumulate in the dead or injured cells [2]. TNV-infected bean hypocotyls represent an example of a progressive interaction between living and dead tissues. The differences in the yields of phytoalexins from virus-infected hypocotyls can thus be explained. A prolonged interaction between living and dead tissues, particularly one in hypocotyls which are physiologically active, e.g. as a result of photosynthesis or due to the retention of cotyledons, would enhance phytoalexin synthesis. If the entire hypocotyl died rapidly or if the metabolic activity of the tissue was low, less phytoalexin would be produced. The precise biochemical nature of the processes leading to greater phytoalexin production were not investigated, but probably
Light and accumulation of phytoalexins
83
include enhanced provision of energy, increased supply of metabolic precursors and possibly changes in concentrations and/or distribution of endogenous hormones [lo]. The differences in phytoalexin concentration observed here would not appear to be due to differences in susceptibility to TNV, since neither light nor the presence of cotyledons had a great effect on the amount of virus produced. In contrast to the other phytoalexins, phaseollinisoflavan was only detected in hypocotyls exposed to light : it was not present in any tissues maintained in complete darikness. The presence of cotyledonous tissue and/or roots on dark-grown seedlings did not lead to production of this phytoalexin. It is unlikely that formation of phaseollinisoflavan was prevented solely by lack of energy supplies since other phytoalexins were synthesized to a limited extent. Synthesis of phaseollinisoflavan may require a specific light-mediated response, perhaps for synthesis of certain enzymes [24]. Whether the reactions converting phaseollin to phaseollinisoflavan [13] are involved is not known. If they are, an explanation must then be sought for the loss of phaseollin which occurred after prolonged incubation in darkness [Fig. 1(a) and (c)l. The importance of light for maximum yields of phaseollinisoflavan is also evident from research with bean leaves. Leaves infected with bacteria contained more than 100 pg phaseollinisoflavan g-i leaf and this concentration was greater than that of phaseollin in the same tissues [12]. l’n summary, the present results demonstrate that the physiological state of bean seedlings can affect their ability to accumulate phytoalexins. In seedlings of legumes, resistance to several pathogenic fungi, e.g. Rhizoctonia solani and Colletotrichum lindemuthianum is due to the accumulation of phytoalexins at concentrations which prevent pathogen development [4, 221. Further investigations are now required to determine whel:her the phytoalexin response of fungus-infected tissues can be modified by physiological factors such as those described above and, if so, whether this can be related to changes in seedling resistance. REFERENCES 1. BAILEY, J. A. (1969). Phytoalexin production by leaves of Pisum s&urn in relation to senescence. Annals of Applied Biology 64, 3 15-324. 2. BAILEY, J. A. (1982). Physiological and biochemical events associated with the expression of resistance to disease. In Active Defence Mechanisms in Plants, Ed. by R. K. S. Wood, pp. 39-65. Plenum Press, London & New York. 3. B.AILEY, J. A. & BURDEN, R. S. (1973). Biochemical changes and phytoalexin accumulation in Pluseolus vulgaris following cellular browning caused by tobacco necrosis virus. Physiological Plant Pathology 3, 171-177. 4. B.ULEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolus vulgaris) and physiological races of Colletotrichum lindemuthianum. Physiological Plant Pathology 1, 435-449. 5. B.ULEY, J. A. & SKIPP, R. A. (1978). Toxicity of phytoalexins. Proceedings of the Association of Applied Biologists. Annals of Applied Biology 89, 354-358. 6. BAILEY, J. A., VINCENT, G. G. & BURDEN, R. S. (1976). Th e antifungal activity of glutinosone and capsidiol and their accumulation in virus-infected tobacco species. Physiological Plant Pathology 8,35-41. 7. BN~IXN, F. C. & HARRISON, B. D. (1955). Stud& on the multiplication of tobacco necrosis virus in inoculated leaves of French bean plants. 3ournal of General Microbiology 13,494-508. 8. BAWDEN, F. C. & ROBERTS, F. M. (1947). The influence of light intensity on the susceptibility of plants to certain viruses. Annals of Applied Biology 34,286-296. 9. CRUICKSHANIC, I. A. M. & PBRRIN, D. R. (1963). Studies on phytoalexins. VI. Pisatin: the effect of some factors on its formation in Pisum sativum L. and the significance of pisatin in disease resistance. Australian 3ournal of Biological Science, 16, 111-128.
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84
10. DE LAAT, A. M. M., BRANDENBURO,D. C. C. & VAN LOON, L. C. (1981). The modulation of the conversion of I-aminocyclopropane-1-carboxylic acid to ethylene by light. Plan& 153, 193-200. 11. &8JARDINS, P. R. (1969). Alfalfa mosaic virus induced lesions on bean, the effect of light and temperature. Plant LXseass Rcpxter 53,30-33. 12. GNANAbfANlCKAM, S. N. & PATIL, S. S. (1977). Accumulation of antibacterial isoflavanoids in hypersensitively responding bean leaf tissues inoculated with Pseudomonas phascolicola. Physiological Plant Pathology 10, 159-168. 13. &RGREAVBS, J. A. & SELBY, C. (1978). Phytoalexin formation in cell suspensions of Phuseolus vulgaris in response to an extract of bean hypocotyls. Phytochemistty 17,1099-l 102. 14. HELLER. W., EDIN-BUHLER, B., GARI)INER, S. E., KNOBLOCH, K. H., MATERN, U., EBEL, J. & HAHLBROCK, K. (1979). Enzymes of general phenylpropanoid metabolism and of flavonoid glycoside biosynthesis in parsley: Differential inducibility by light during the growth of cell suspension cultures. Plant Physiology 64, 371-373. 15. ti, W. C. (1967). The effect of darkening on the susceptibility of French bean to tobacco necrosis virus. Canadian Journalof Botany 45,543-553. 16. KL~N, W. L. & HAMMERSCHLAG, F. (1972). Production of the phytoalexin, hydroxyphaseollin, in soybean leaves inoculated with tobacco necrosis virus. Phytojablogy 62,7 19-72 1. 17. LAMPARD, J. F. (1974). Demethylhomopterocarpin: an antifungal compound in Canavaliu etzsifwmis and Vigna unguiculata following infection. Phytochcmisby 16, 1193-l 196. 18. MURCH, R. S. & PAXTON, J, D. (1977). Glyceollin concentrations in Phytophthora resistant soybean: Light influences. Annual Meeting Abstracts 247. Proceedings of the American Phytojathological sociegv4. 19. MUR~H, R. S. SKPAXTON, J. D. (1980). Temperature and glyceollin accumulation in Phy~tophthorresistant soybeans. Phytopathologische ,Qitschr$t 97,282-285. 20. RAHE, J. E. & ARNO~, R. M. (1975). Injury-related phaseollin accumulation in Phaseolus vulguris and its implications with regard to specificity of host-parasite interaction. Canadian 3ournal of Botany
53,921-928.
21. ROWELL, P. M. (1981). Studies on the production of phytoalexins by plant tissues and their importance in resistance of Phaseolus vulgaris to fungal disease. M.Phil. thesis, University of London. 22. SMITH, D. A., VAN ETXZN, H. D. & BATEMAN, D. E. (1975). Accumulation of phytoalexins in Phaseolus vulgaris hypocotyls following infection with Rhizoctonia solani. Physiological Plant P&olo~
5,51-64.
23. UEOAKX, R., FUJIMORI, T., Kuao, S. & KATO, K. (1981). Sesquiterpenoid stress compounds from Ncotiana species. Phy~ochemistry 29, 1567-1568. 24. WARD, E. W. B., STOGSEL, P. & LAZAROVITS, G. (1981). Similarities between age-related and race-specific resistance of soybean hypocotyls to Phytophthora megasperma var. sojac. Phy~ojxathologp 71,504-508.
Influence of light and excision of organs on accumulation of phytoalexins in virus-infected hypocotyls of bean (Phaseohs vulgaris) P.
M.
ROWELL
and J. A. BAILEY
Long Ashton ResearchStation, University of Bristol,
Long Ashton,
Bristol BS18 9AF, U.K.
(Accq!&dforpublication March 1982)
The phytoalexins, phaseollin, phaseollidin, phaseollinisolIavan and kievitone, were isolated from bean hypocotyls bearing TNV-induced necrotic lesions. Phaseollin was the predominant phytoalexin and was localized within the necrotic tissues. Highest yields of phase&n (more than 450 pg g-1 fresh wt) were obtained from dark-grown infected bean seedlings brought into light. The production of each phytoalexin was reduced when cotyledons were removed. Gxnvth and incubation in total darkness completely prevented the production of any phaseollinisoflavan. The total amount of virus produced was not greatly affected by these treatments. It is suggested that the amounts of phytoalexin which accumulate depend on the different biochemical and physiological activities of the living tissues with which adjacent necrotic tissues interact.
INTRODUCTION Phytoalexins accumulate in plants in response to injurious stimuli [Z], including infections by viruses which cause formation of local necrotic lesions. Studies have been reported with virus-infected Phaseolus vulgaris, Glycine max, Pisum sativum, Vigna sinenrti, Canavalia ensiformis and several species of Nicotiana [2, 6, 16, 17, 231. Four phytoalexins, phaseollin, phaseollidin, phaseollinisoflavan and kievitone, were isolated from tobacco necrosis virus (TNV)-infixted bean hypocotyls [3]. This tissue was later used as a source of the phytoaiexins for studies on their metabolism ‘by fungi and plants and for assessing their antimicrobial activity [5]. Yields of phytoalexins from TNV-infected hypocotyls varied considerably, from O-01 to 2 mg g -I tissue. The present investigation was aimed at defining optimum conditions for the accumulation of phytoalexins in bean seedlings. Subsequently the influence of light and the relative contribution of leaves, roots and cotyledons were studied. MATERIALS AND METHODS Virus and plant material Tobalcco necrosis virus was obtained as described previously [3]. However, on this occasion the virus suspension was prepared from living rather than frozen tissue, since the virus titre was found to be much greater in living tissues than in those stored1 at -20 “C. All seedlings were grown in complete darkness from seeds of Phaseoh vulgaris, cv. Prince, sown in seed trays containing sterilized vermiculite which was kept moist 0048-4059/82/040075
+ 10 $03.00/O
@
1982 Academic
Press
Inc.
(London)
Limited
76
P. M. Rowell
and J. A. Bailey
by adding tap water only. Darkness was ensured by placing the trays, immediately after seed sowing, in boxes which totally excluded light in a dark room protected by a light trap. They were incubated at 22&l “C. Inoculation and incubation of bean hypocotyl tissue Hypocotyls (15 to 20 cm long) of 7-day-old seedlings were inoculated by rubbing with foam sponge soaked with a suspension of TNV containing Carborundum; The virus titre was adjusted to give browning throughout the hypocotyls without causing the tissues to wilt or desiccate. Inoculations were carried out under green light and within 5 min the hypocotyls were washed with water. Inoculated seedlings were incubated intact or after excision of various tissues. All cut surfaces were sealed with petroleum jelly. Seedlings lacking roots and excised hypocotyls were placed on their sides in transparent plastic boxes; those retaining their roots remained in the vermiculite. Inoculated tissues were incubated at 22fl “C, either in total darkness (see above) or in a growth room illuminated for periods of 16 h with fluorescent light (8 to 10 klx) followed by 8 h darkness. ” Measurement of phytoalexin concentration Sections (10 cm) cut from the middle of inoculated hypocotyls were used for analysis of phytoalexins; Each sample usually consisted of 7 sections, each weighing approximately 1 g. Phytoalexins were obtained by methods based on those used previously [4]. Tissue was macerated in ethanol and after evaporation the aqueous phase was partitioned with di-ethyl ether. Initial separation of the ether extract was achieved on silica thin layers (Merck 5715, 0.25 mm) using chloroform and ethanol (100 : 4, v/v). This solvent led to purification of phaseollin, phaseollidin and phaseollinisoflavan. Kievitone was purified by subsequent repeated development with chloroform and ethanol (100 : 5). The phytoalexins were eluted from the silica with ethanol and their concentrations measured by ultraviolet spectroscopy. Losses during purification were estimated to be approximately 25% for phaseollin, phaseollidin and phaseollinisoflavan and 35% for kievitone. Diazotized p-nitroaniline (DpNA) was used to detect trace amounts of phytoalexins on chromatograms. The final concentrations of phytoalexins are usually expressed in terms of their weight per hypocotyl. This indicates total yield of phytoalexin and avoids problems associated with loss of tissue weight (dehydration) during later stages of symptom development. Measureementof virus titre Two sections (10 cm) of hypocotyl tissue were macerated in 2 ml of distilled water. The mixture was filtered through muslin and a little Carborundum was added to the filtrate. The filtrate was used to inoculate the 3rd and 4th leaves of two, 8 to lo-weekold plants of Ncotiana tabacum cv. White Burley. The average number of lesions on half of each leaf was recorded 8 days later. RESULTS
Seedlings grown in total darkness for 7 days consisted of a white hypocotyl, 15 to 20 cm long, with small pale yellow leaves contained between the cotyledons. Continued incubation of seedlings in darkness led to extensive elongation of the epicotyl (10 to
Light and accumulation of phytoalexins
77
20 cm) but the leaves remained small and did not expand greatly; there was no green pigmentation. When transferred to light, all the tissues of the seedlings turned green; the epicotyl remained short (less than 5 cm) and the leaves expanded. Excised hypocotyls, i.e. lacking cotyledons, appeared identical when kept in darkness or transferred to light, showing a complete lack of green pigments. Hypocotyls bearing cotyledons always turned green when placed in light. Effect
of light
on accumulation
of phytoalexinr
In darkness, discrete brown lesions were first observed 32 to 37 h after inoculation on both intact seedlings and on excised hypocotyl sections. In light, brown lesions did not develop until 10 to 12 h later. Further incubation led to increased browning, until the entire hypocotyl tissues were discoloured. As in previous investigations [3], the production of phytoalexins was closely associated with symptom development and phaseollin was concentrated within the lesion tissue (Table 1). Only traces of this phytoalexin were detected in tissue immediately surrounding the lesions and none could be found in the unaffected tissue between the lesions. Thin layer chromatograms of these extracts indicated that phaseollidin and kievitonewere distributed similarly. TABLE
Distribution
Tissue
of phaseollin
hypoco@ls of intact seedlings
Tissue Wt (9)
extracted
Necrotic lesions Tissute adjacent to necrotic Remaining tissue Entire hypocotyls
!
in TM’-infected
tissue
1.21 1.61 1.41 1.93
Amount (I.@) 195 t n.d. 96
of phaseollin (f.tg g-r tissue) 174 t n.d. 50
Seven-day-old dark-grown seedlings were inoculated with TNi and incubated in darkness for 3 days, at which time discrete superficial lesions were present. t, Phaseollin was detected on t.1.c. plates with DpNA: amount present was less than 5 ug; nd., phaseollin was not detected with DpNA: limit of detection was less than 0.5 pg.
A, comparison of phytoalexin concentrations in intact seedlings and in excised hypocotyl sections incubated in light or darkness is illustrated in Fig. 1. Phaseollin accumulated more quickly during the initial stages of symptom development in hypocotyls incubated in darkness [Fig. 1 (a) and (c)] than in light [Fig. 1(b) and (d)]. In darkness, the concentrations of all phytoalexins, in both intact seedlings and excised hypocotyl sections, increased to a maximum after 6 to 8 days, when hypocotyls of intact seedlings contained approximately twice as much of each phytoalexin as excised sections. After 8 days slight reductions in phytoalexin concentrations occurred. In light, the accumulation of phaseollidin and kievitone in both intact seedhngs and excised hypocotyls followed a pattern similar to that found in darkness, although kievitone appeared to be produced earlier in excised hypocotyls. The major effect of light was observed with intact seedlings, where the production of phase:ollin continued for more than 8 days, such that after nearly 10 days the yield of phase:ollin exceeded 450 pg per hypocotyl; a concentration which was more than 6 times that obtained from excised hypocotyls incubated in light and, as above, was
P. M. Rowell end J. A. Bailey
78
(d)
6 Perlod
of incubotlon
8
(h 1
FIG. 1. Accumulation of phytoalexins in TNV-infected hypocotyl tissue. Hypocotyls of dark-grown bean seedlings were inoculated with TNV. Intact seedlings or excised hypocotyl sections were incubated in light or darkness. Phytoalexin concentrations in hypocotyl tissue (b) Hypocotyls were measured at intervals. (a) Hypocotyls of seedlings incubated in darkness. of seedlings incubated in light. (c) Excised hypocotyl sections incubated in darkness. (d) Excised hypocotyl sections incubated in light. 0, Phaseollin; A, phaseollidin; X, kicvitonc; 0, phaseollinisoflavan.
Light and accumulation of phytoalexins
79
twice that obtained from intact seedlings incubated in darkness. In contrast, however, excised hypocotyl sections produced 75 jtg phaseollin per hypocotyl when incubated in light and this was less than that found in excised sections maintained in darkness (129 pg per hypocotyl). In all these experiments the maximum concentrations of phaseollidin and kievitone were less than those of phaseollin, varying between 19 and 86 l,tg per hypocotyl. Phaseollinisoflavan was not detected in tissues maintained in complete darkness. Aftler incubation in light this phytoalexin was present in trace amounts in excised hypocotyl sections, and in greater quantities (50 pg per hypocotyl) in entire seedlings, especially when incubated for a long time (9 to 14 days). Con,tribution of cotyledons,leavesand roots to production of phytoalexins The previous results indicate that hypocotyls of intact seedlings produce greater quantities of phaseollin when incubated in light than in darkness and that, in light, inta.ct seedlings produce much more phaseollin than excised hypocotyl sections. The following experiments assessedthe contribution of cotyledons, leaves and/or roots to the accumulation of phytoalexins in bean hypocotyls. Hypocotyls were inoculated with TNV, the appropriate organs were removed and the remaining tissue incubated in 1:ight or darkness for 7 days. No differences in the time of symptom appearance were observed with hypocotyls of intact seedlings and those lacking cotyledons, roots and/or leaves. The results are presented in Tables 2 and 3. As in the earlier experiments (see Fig. l), the amounts of phytoalexin produced in tissues incubated in light or darkness did not differ greatly at the 7 day sampling stage. :Removing both cotyledons caused substantial reductions, to between one-half and one-fifth, in the concentrations of phaseollin and phaseollidin produced in either light or darkness (Table 2). Similar reductions occurred if the leaves were also Eff&
Tissue
of removing
removed
None Two cotyledons All leaves Two cotyledons and all leaves One ~cotyledon Two half cotyledons One and a halfcotyledons Two, threequarter cotyledons One and a threequarter cotyledons
cotyledonous
TABLE 2 tissue on the accumulation seea%&
Phytoalexin Phaseollin Dark Light
of phytoalexins
in hypOcolylE
of
bean
concentration (ug/hypocotyl) Phaseollidin Phaseoilinisoflavan Dark Light Dark Light 73.0*2.0 23.0&3.0 735*0*5
69-O&9.0 30.5*0.5 86*5*7.5
n.d. n.d. n.d.
11.5*0*5 4-F 19.0’
89.555.5 85.5& 14.5 184*0&4.0 208.0*13*0 136.5&l 1.5 1965*26.6
27.055.0 65.5&6.5 46.0 *9-O
36.563.5 76*5+8-5 72.5 ho.5
n.d. n.d. nd.
3.0+ 1.0 10*0*0 ll*o*l~o
135*5+7.5
179.0*22.1
54.0&10.0
67.0&15.0
n-d.
8.0*
1.0
121.542.5
174.5+25.6
41.0+6.0
64.0&
n.d.
7*0*
1.0
9O.Ok5.0
171.5*31*6
35.54
n.d.
7*0*1*0
205.0&8*0 93*5*2-5 207.Ok9.0
259.5 i30.6 141.5*14.5 231.0*29*1
1.5
59-O&6.0
14.0
n.d.. Phaseollinisoflavan not detected with DpNA : limit of detection was less than 0.5 fig. Se&day-old dark-grown seedlings were inoculated with TNV. Cotyledonous &sue was removed, and the seedlingsincubated in light or darkness. Samples of 7 hypoeetyls sections were extracted after 8 days. Values are means and standard errors of 2 experiments, except where indicated.U
80
P. M. Row4 and J. A. Bailey
removed but removal of the leaves alone caused no effect. Phaseollinisoflavan was again not detected in any tissues incubated in darkness. It was present in all tissue incubated in light, but removing cotyledons reduced production of phaseollinisoflavan to one-quarter. Accumulation of phaseollinisoflavan was greatest in seedlings lacking leaves ; an effect which was observed in several other experiments [ZO]. The contributions of different amounts of cotyledonous tissue to the accumulation of phytoalexins are also presented in Table 2. In darkness, removal of increasing portions of cotyledonous tissue caused a corresponding reduction in the concentrations of phaseollin and phaseollidin which formed. This correlation was not observed in seedlings incubated in light; provided a small portion of one cotyledon remained intact, the quantity of phytoalexin produced remained constant, at between 70 to 75% of that found in intact seedlings. Removing roots had very little effect on the amounts of phytoalexin produced (Table 3). TABLE
Effect of removal of roots and other organs on accumlaGon
3
of phaseollin in hypootyls of beansedlings Phase&n concentration Dar~ hyPocoW-l)
Tissue None Roots Roots Roots Roots,
removed
and cotyledons and leaves cotyledons and leaves
Light 193*5* 10-5 206-O& 14.0 73*0*13*0 15&O&26.1 63.5hl5.6
201*5*27-6 192*0*15.0 100*5*15.0 190.5k22.8 85*5&4-5
Seven-day-old dark grown seedlings were inoculated with TNV. Roots, cotyledons and/or leaves were removed, cut surfaces sealed with petroleum jelly and the remaining tissue was incubated in light or darkness. Samples of 7 hypocotyl sections were extracted after 8 days. The values are means and standard errors of 2 separate experiments.
Effect of physiological factors on virus multiplication The number of infective virus particles in hypocotyl tissues inoculated with TNV and incubated in darkness or light is shown in Table 4, which also reports the amounts of phaseollin produced. Intact seedlings maintained constant titres for at least 7 days. Removing cotyledons and/or leaves did not effect the virus titre, but lack of roots, cotyledons and leaves caped a reduction in titre after incubation for 7 days. Differences in phaseollin concentration showed similar trends to those found in earlier experiments. No relationship was apparent between virus titre and phaseollin accumulation.
DISCUSSION Prior to inoculation with TNV, all bean seedlings were grown in complete darkness. This ensured that the hypocotyl tissue was susceptible to virus infection and replication [7, 8, II, 151, but more importantly it allowed an assessment of the influence of light on the subsequent accumulation of phytoalexins.
two
Of
and
40.3hl.8 -
34.8k3.1
Dark
3 Days
h+opl
34.8*5.9 -
26.3 k4.0
Light
incubated
T-U 4 in light or dknkess
20.8-14.1 -
37~3*5.6
leaf
17.0&2.7 36.3k5.7 39.3 54.3 36.8A4.1
34.3h5.5
Dark
14.8k2.8 40.3 f 7.6 35.Q 1.8 33.8k4.0
42.553.3
Light
and/or
leaves were removed, taken for measurement
7 Days
and after removal of roots, cotyledons
were inoculated with TNV. Roots, cotyledons and/or incubated in light or darkness. Samples of tissue were
33.3+2.8 -
35.3s3.8
Number of lesions per half of &otiana tab&m 5 Days Dark Light
tissue of seedlings
Hypocotyls of 7-day-old dark grown seedlings with petroleum jelly and the remaining tissue phaseollm concentrations.
and
cotyledons
TAT-incubakd
leaves
virus from
removed
Two cotyledons Leaves Two cotyledons
IfZlVeS
None Roots,
Tissue
Ti&e
concentration 7 Days
83 84 132 63
163
Light
cut surfaces sealed of virus titre and
87 81 145 77
182
Dark
his bwcovl - ‘1
Phase&in
leaves
82
P. M. Rowell and J. A. Bailey
The influence of host physiology on production of phytoalexins has not received much attention, although greater physiological activity, e.g. as found in younger rather than senescent tissues [I], in tissues incubated at higher temperatures [19], in high oxygen concentrations [9] or in light [18], leads to increased accumulation of phytoalexins. In the present experiments, greatest yields of each phytoalexin were obtained from hypocotyls of intact seedlings incubated in light. High concentrations resulted from applying a virus suspension that caused the entire tissue to become brown but allowed it to remain turgid for a long time. Under these conditions a period of between 7 and 10 days was needed for maximum yields, and slight reductions in the concentrations of phaseollin, phaseollidin and kievitone occurred when seedlings were incubated longer. If the virus suspension was more concentrated, yields of phytoalexin were much reduced due to early wilting and desiccation of the entire hypocotyl [21]. When the contributions of individual factors responsible for the high yields of phytoalexins were studied, it was found that incubation in light with cotyledons remaining intact produced greatest yields. Light also increased accumulation of glyceollins in soybean seedlings infected with Phytophthora megasperma [l&j. In excised bean hypocotyl sections, however, less phytoalexin was produced in light than in darkness. This may be associated with delayed symptom expression such that phytoalexin synthesis occurs when the biochemical reserves of the tisuse have been reduced. The presence of both cotyledons led to greatest accumulation of phytoalexins in the hypocotyls. In darkness removing increasing amounts of cotyledonous tissue produced a progressive decline in the amount of phytoalexin produced. Thus the presence of a quarter of one cotyledon increased phytoalexin accumulation only slightly. In light, however, a quarter of one cotyledon was sufficient to double the yield found in seedlings lacking both cotyledons. The presence or absence of roots did not much affect yield. This contrasts with studies on production of phaseollin in wounded bean seedlings by Rahe & Arnold [20], which showed that removing roots decreased phytoalexin formation. Research with both bean and soybean hypocotyls has also shown that tissue adjacent to the roots produces more phytoalexin than tissue adjacent to the cotyledons [20,24]. In the present study such effects were avoided by excising tissue from the centre of the etiolated hypocotyls. The close association between cell death and accumulation of phytoalexins was discussed previously [2] and was confirmed in the present work. It has been proposed that phytoalexins accumulate in tissues as a consequence of localized cell injury which initiates an interaction between injured and adjacent living cells. This interaction leads to synthesis of phytoalexins in the living tissue from where they diffuse to accumulate in the dead or injured cells [2]. TNV-infected bean hypocotyls represent an example of a progressive interaction between living and dead tissues. The differences in the yields of phytoalexins from virus-infected hypocotyls can thus be explained. A prolonged interaction between living and dead tissues, particularly one in hypocotyls which are physiologically active, e.g. as a result of photosynthesis or due to the retention of cotyledons, would enhance phytoalexin synthesis. If the entire hypocotyl died rapidly or if the metabolic activity of the tissue was low, less phytoalexin would be produced. The precise biochemical nature of the processes leading to greater phytoalexin production were not investigated, but probably
Light and accumulation of phytoalexins
83
include enhanced provision of energy, increased supply of metabolic precursors and possibly changes in concentrations and/or distribution of endogenous hormones [lo]. The differences in phytoalexin concentration observed here would not appear to be due to differences in susceptibility to TNV, since neither light nor the presence of cotyledons had a great effect on the amount of virus produced. In contrast to the other phytoalexins, phaseollinisoflavan was only detected in hypocotyls exposed to light : it was not present in any tissues maintained in complete darikness. The presence of cotyledonous tissue and/or roots on dark-grown seedlings did not lead to production of this phytoalexin. It is unlikely that formation of phaseollinisoflavan was prevented solely by lack of energy supplies since other phytoalexins were synthesized to a limited extent. Synthesis of phaseollinisoflavan may require a specific light-mediated response, perhaps for synthesis of certain enzymes [24]. Whether the reactions converting phaseollin to phaseollinisoflavan [13] are involved is not known. If they are, an explanation must then be sought for the loss of phaseollin which occurred after prolonged incubation in darkness [Fig. 1(a) and (c)l. The importance of light for maximum yields of phaseollinisoflavan is also evident from research with bean leaves. Leaves infected with bacteria contained more than 100 pg phaseollinisoflavan g-i leaf and this concentration was greater than that of phaseollin in the same tissues [12]. l’n summary, the present results demonstrate that the physiological state of bean seedlings can affect their ability to accumulate phytoalexins. In seedlings of legumes, resistance to several pathogenic fungi, e.g. Rhizoctonia solani and Colletotrichum lindemuthianum is due to the accumulation of phytoalexins at concentrations which prevent pathogen development [4, 221. Further investigations are now required to determine whel:her the phytoalexin response of fungus-infected tissues can be modified by physiological factors such as those described above and, if so, whether this can be related to changes in seedling resistance. REFERENCES 1. BAILEY, J. A. (1969). Phytoalexin production by leaves of Pisum s&urn in relation to senescence. Annals of Applied Biology 64, 3 15-324. 2. BAILEY, J. A. (1982). Physiological and biochemical events associated with the expression of resistance to disease. In Active Defence Mechanisms in Plants, Ed. by R. K. S. Wood, pp. 39-65. Plenum Press, London & New York. 3. B.AILEY, J. A. & BURDEN, R. S. (1973). Biochemical changes and phytoalexin accumulation in Pluseolus vulgaris following cellular browning caused by tobacco necrosis virus. Physiological Plant Pathology 3, 171-177. 4. B.ULEY, J. A. & DEVERALL, B. J. (1971). Formation and activity of phaseollin in the interaction between bean hypocotyls (Phaseolus vulgaris) and physiological races of Colletotrichum lindemuthianum. Physiological Plant Pathology 1, 435-449. 5. B.ULEY, J. A. & SKIPP, R. A. (1978). Toxicity of phytoalexins. Proceedings of the Association of Applied Biologists. Annals of Applied Biology 89, 354-358. 6. BAILEY, J. A., VINCENT, G. G. & BURDEN, R. S. (1976). Th e antifungal activity of glutinosone and capsidiol and their accumulation in virus-infected tobacco species. Physiological Plant Pathology 8,35-41. 7. BN~IXN, F. C. & HARRISON, B. D. (1955). Stud& on the multiplication of tobacco necrosis virus in inoculated leaves of French bean plants. 3ournal of General Microbiology 13,494-508. 8. BAWDEN, F. C. & ROBERTS, F. M. (1947). The influence of light intensity on the susceptibility of plants to certain viruses. Annals of Applied Biology 34,286-296. 9. CRUICKSHANIC, I. A. M. & PBRRIN, D. R. (1963). Studies on phytoalexins. VI. Pisatin: the effect of some factors on its formation in Pisum sativum L. and the significance of pisatin in disease resistance. Australian 3ournal of Biological Science, 16, 111-128.
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84
10. DE LAAT, A. M. M., BRANDENBURO,D. C. C. & VAN LOON, L. C. (1981). The modulation of the conversion of I-aminocyclopropane-1-carboxylic acid to ethylene by light. Plan& 153, 193-200. 11. &8JARDINS, P. R. (1969). Alfalfa mosaic virus induced lesions on bean, the effect of light and temperature. Plant LXseass Rcpxter 53,30-33. 12. GNANAbfANlCKAM, S. N. & PATIL, S. S. (1977). Accumulation of antibacterial isoflavanoids in hypersensitively responding bean leaf tissues inoculated with Pseudomonas phascolicola. Physiological Plant Pathology 10, 159-168. 13. &RGREAVBS, J. A. & SELBY, C. (1978). Phytoalexin formation in cell suspensions of Phuseolus vulgaris in response to an extract of bean hypocotyls. Phytochemistty 17,1099-l 102. 14. HELLER. W., EDIN-BUHLER, B., GARI)INER, S. E., KNOBLOCH, K. H., MATERN, U., EBEL, J. & HAHLBROCK, K. (1979). Enzymes of general phenylpropanoid metabolism and of flavonoid glycoside biosynthesis in parsley: Differential inducibility by light during the growth of cell suspension cultures. Plant Physiology 64, 371-373. 15. ti, W. C. (1967). The effect of darkening on the susceptibility of French bean to tobacco necrosis virus. Canadian Journalof Botany 45,543-553. 16. KL~N, W. L. & HAMMERSCHLAG, F. (1972). Production of the phytoalexin, hydroxyphaseollin, in soybean leaves inoculated with tobacco necrosis virus. Phytojablogy 62,7 19-72 1. 17. LAMPARD, J. F. (1974). Demethylhomopterocarpin: an antifungal compound in Canavaliu etzsifwmis and Vigna unguiculata following infection. Phytochcmisby 16, 1193-l 196. 18. MURCH, R. S. & PAXTON, J, D. (1977). Glyceollin concentrations in Phytophthora resistant soybean: Light influences. Annual Meeting Abstracts 247. Proceedings of the American Phytojathological sociegv4. 19. MUR~H, R. S. SKPAXTON, J. D. (1980). Temperature and glyceollin accumulation in Phy~tophthorresistant soybeans. Phytopathologische ,Qitschr$t 97,282-285. 20. RAHE, J. E. & ARNO~, R. M. (1975). Injury-related phaseollin accumulation in Phaseolus vulguris and its implications with regard to specificity of host-parasite interaction. Canadian 3ournal of Botany
53,921-928.
21. ROWELL, P. M. (1981). Studies on the production of phytoalexins by plant tissues and their importance in resistance of Phaseolus vulgaris to fungal disease. M.Phil. thesis, University of London. 22. SMITH, D. A., VAN ETXZN, H. D. & BATEMAN, D. E. (1975). Accumulation of phytoalexins in Phaseolus vulgaris hypocotyls following infection with Rhizoctonia solani. Physiological Plant P&olo~
5,51-64.
23. UEOAKX, R., FUJIMORI, T., Kuao, S. & KATO, K. (1981). Sesquiterpenoid stress compounds from Ncotiana species. Phy~ochemistry 29, 1567-1568. 24. WARD, E. W. B., STOGSEL, P. & LAZAROVITS, G. (1981). Similarities between age-related and race-specific resistance of soybean hypocotyls to Phytophthora megasperma var. sojac. Phy~ojxathologp 71,504-508.