Phytoalexin involvement in the latent infection of Capsicum annuum L. fruit by Glomerella cingulata (Stonem.)

Physiological Plant Pathology (1982) 21, 161-170

Phytoalexin involvement in the latent infection of Capsicum annuum L. fruit by Glomerella cingulata (Stonem.) N.

K.

B. ADIKARAM,

AVERIL E. BROWN and T. R. SWINBURNE~

Plaut Pathology ResearchDivision, Department of Agriculture for Jvorthern Ireland and Facul& of Agriculture and Food Science,Queen’s University, Belfast, U.K. (Acceptedfor publication May 1982)

Immature Caps&am aaautun L. fruits wound-inoculated with conidia of Glomerella cingukata (Stonem.) did not develop progressive lesions until fruit ripening was well advanced. Juice expressed from inoculated tissue was inhibitory to G. cingulata. Thin layer chromatography (t.1.c.) of ether extracts of inoculated tissue, bioassayed with G. cingulata or Clados@+mz, demonstrated the presence of one prominent inhibition zone. This fungitoxic compound, capsicannol, began to accumulate within 18 h after inoculation in the immature fruit tissue, reached maximum concentrations after 4 days and was more concentrated in superficial than in deeper tissues. Capsicannol and cap&Sol, which accumulated in inoculated ripening fruit tissue, had declined to non-toxic levels at the onset of progressive lesion development. Diminution of the 2 phytoalexins was accompanied by the appearance of capsenone and another compound which showed slight fimgitoxicity. Capsidiol and capsicannol, which accumulated in tissue treated with an elicitor from G. cingulata mycelial walls, declined at a rate similar to that in tissue inoculated with conidia, and both compounds were degraded in liquid culture by G, cingulata.

INTRODUCTION

of Capsicum annuum fruits, generally known as anthracnose of peppers, develops from latent infections in immature fruits. Conidia of Glomerella cingulata germinated on the surface of uninjured or injured immature fruits and produced appressoria. A small proportion of the appressoria produced thin infection hyphae which penetrated the cuticle and outer epidermal cell wall of the immature fruits, then ceased to grow most remaining dormant as latent appressoria until fruit ripening commenced [Z] . Latent infection in bananas caused by Colletotrichum musae has been attributed, at least in part, to the accumulation of antifungal compounds [a. Two types of appressoria are produced by C. murae; one thin walled and hyaline, the other thick walled and dark [IO]. Early necrosis [IO] and phytoalexin accumulation [Is], following inoculation of green fruits, was found to be associated with the early development of penetration hyphae from the hyaline appressoria. These penetration hyphae failed to develop further and the colonization of ripening fruit began with the much later development of penetration hyphae from the dark appressoria. Riperot

f Present address: Crop Protection ME19 6BJ, U.K. 0048/4059/82/050161+ 10 $03.00/O

Division,

East Malling

Research Station,

Maidstone,

@ 1982 Academic Press Inc. (London)

Kent

Limited

162

N. K. 0. Adikaram

et al.

Stoessl, Unwin & Ward [11] isolated a sesquiterpenoid phytoalexin, capsidiol, from the diffusates of ripening fruits of Capicum frutescent L. (C. annuum L.) produced in response to infection by several fungi, some of which were pathogenic and others non-pathogenic. The significance of capsidiol accumulation in several compatible and incompatible interactions between ripening fruits and fungi has been assessed[7, 8, 12, 13, 16, 171. The role of phytoalexins in the resistance of immature C. annuum fruits to anthracnose fungi has not been studied extensively. In the experiments reported here the involvement of post-infectionally formed antifungal compounds in the development of latent infections in immature C. annuum fruits was investigated. MATERIALS

AND

METHODS

Fruits Fruits of C. annuum var. FI Belboy were obtained from plants grown in a glasshouse at 2055 “C with a 17 h photoperiod. Fruits were harvested for use in experiments 35 days after pollination when they were green and immature but had reached maximum size. Green, immature fruits of unknown variety obtained from the local market were also used in some experiments. Fruits were allowed to ripen in moistened chambers in the laboratory at 2Oh2 “C. Ripening and fully ripened fruits were obtained after 10 and 30 days storage, respectively. Fungus G. cingulata (IMI 85093) used in the experiments was maintained on Cook’s No. 2 medium containing a mineral supplement [14]. Pathogenicity was maintained by inoculation and reisolation from C. annuum fruit at monthly intervals. Suspensions of conidia were prepared in sterile distilled water from 5-day-old cultures. Conidia were washed 3 times by centrifugation and resuspension. Inoculation Fruits, free from damage of any kind, were selected, wiped with methanol and allowed to dry. The peel was punctured with a 3 mm long sterile needle attached to a glass rod, 16 to 25 punctures being made per fruit depending on the size of the fruit. Drops (20 pl) of suspensions (5 X IO5 ml-l) of washed conidia were applied to the punctured sites or to the unpunctured surface of fruit. Control fruits were treated with drops (20 ~1) of sterile distilled water. When large quantities of inoculated tissue were required, the upper surfaces of fruits were extensively punctured and sprayed with a suspension (5 X lo5 ml-l) of conidia or sterile distilled water using a Gazjet sprayer. The inoculated and control fruits were supported on glass tubes in moist chambers and held at 20&2 “C. To obtain diffusates, 20 ml aliquots of suspensions (5 x lo5 ml-r) of conidia were injected into cavities of excised fruits. The fruits were held in moist chambers for 44 h at 20-&2 “C, then cut open and the diffusates collected [II]. Preparation of tissuefor bioassayand solvent extraction When the individual spore drop technique was employed, 6 mm diameter and 3 mm deep discs of tissue were cut from the inoculated regions using a cork borer. In fruits sprayed with suspensions of conidia the pericarp was cut into small pieces, after

Post-infectionally

formed antifungal

compounds

163

discarding the seeds, the inner fleshy parts and the stalk-end. The cut tissue was stored at - 18 “C. Tissue which was to be solvent extracted was freeze dried (Edwards Modulyo freeze drier) at -60 “C and 0.2 atm pressure for 18 h. Juice to be bioassayed was prepared from frozen tissue discs, by squeezing through muslin. The crude juice was clarified by centrifugation (2300 g for 15 min). Solvent extraction, chromatographyand bioassayof phytoalexins Tissue cut from inoculated and control fruits was extracted 3 times in diethyl ether (2 ml g-1 fresh tissue). The ether extracts from each treatment were combined, dried over anhydrous NasSO, and the solvent removed under vacuum at 45 “C. The residue was taken up in chloroform (residue from 1 g of fresh tissue in 1 ml chloroform). Aliquots (O-1 ml) were spotted on grooved t.1.c. plates (M. & B. Chroma-lay ; 100 x 200 mm) with silica gel (Kieselgel60 PF,,,; MERCK; O-25 m m thick) as the stationary phase. The plates were developed in solvent A (n-butanol : ethyl acetate : acetic acid : 5 : 95 : O-1). To detect phytoalexins the plates were air dried and sprayed with a dense suspension of conidia of either Cladosporium cladosporiod8.sor G. cingulata in Czapek-Dox nutrient solution and incubated for 3 days. Compounds toxic to the test fungi were located by the absence of aerial mycelium [9]. For partial purification of phytoalexins, silica gel removed from zones on chromatograms at the RF values of the fungitoxic compounds was finely ground, eluted with chloroform and the chloroform removed under vacuum. The phytoalexins capsidiol, capsenone and a previously unreported compound, capsicannol, were further purified as described by Adikaram et al. [4j. Estimation of concentrationsof phytoalexins in inoculatedfruit tissue To estimate capsicannol concentrations, O-1 ml aliquots of ether extracts from inoculated and control fruit tissue were applied to t.1.c. plates. Samples (0.1 ml) of a dilution series of purified capsicannol were applied to separate t.1.c. plates. The plates were developed in solvent A, air dried and assayed as previously described with C. cladosporiodes.Approximate concentrations of capsicannol present in tissue extracts were determined by comparing the size of inhibition zones with the size of zones produced by known concentrations of the compound. Concentrations of capsidiol and capsenone were measured by g.1.c. in a Hewlett Packard Model 5750G instrument as described by Stoessl, Unwin & Ward [I,?]. Ahquots (5 ~1) of concentrated ether extracts of diffusates of inoculated tissue were injected into the column and the area under the peaks on the chromatograms was measured. Calibration curves for capsidiol and capsenone were constructed with values obtained with a concentration series of the pure compounds. Concentrations of the phytoalexins were determined as mg g-r fresh tissue. Assayfor toxicity of phytoalexins to germination of conidia of G. cingulata Conidia of G. cingulata germinated poorly in sterile distilled water; however, good germination was obtained in juice expressed fkom healthy immature fruit tissue. Dilution series of capsicannol and capsidiol were made in 1% crude juice from immature fruit (1 ml) and a suspension (50 u.l) of conidia (loo ml-l) in sterile distilled water was added to each solution. Drops (20 fl) from each test solution

164

N. K. B. Adikaram

et a/.

were placed on clean glass slides and the slides incubated in moist chambers at 20 “C for 7 h. Two hundred randomly selected conidia from each drop were then counted for germination. Two drops were used per treatment and the experiment was replicated 3 times. Seededcup-plate assay Wells (8 mm diameter) cut in 50% Cook’s No. 2 agar seeded with conidia of G. cingulata were filled (0.1 ml) with juice expressed from test tissue samples. The plates were incubated for 3 days at 20 “C and the diameters of zones of inhibition were measured. Extraction of cell wall comfionentsof G. cingulata which elicit $hytoaIexin production in C. annuum fruits Mycelial discs of G. cingulata were aseptically transferred into Cook’s No. 2 liquid cultures and incubated in an orbital shaker (80 r min-l) for 8 days at 22 “C. Mycelial mats were separated by filtering through Whatman No. 1 paper. Polysaccharides were extracted from the mycelial walls as described by Anderson & Albersheim [5]. To test for elicitor activity, drops (20 pl) of mycelial extracts were applied to wounded or unwounded surfaces of immature or ripe fruits. Control fruits were treated with drops (20 @) of sterile distilled water and all the fruits were incubated in moist chambers at 20 “C. Discs (6 mm diameter) of tissue were removed, extracted in ether and bioassayed or stored at - 18 “C for chemical assay. Degradation of phytoalexins in vitro by G. cingulata A synthetic medium for shake cultures of G. cingulata was prepared as described by Stoessl, Unwin & Ward [I,?]. The medium (10 ml) was dispensed in boiling tubes and autoclaved at 121 “C for 20 min. Aliquots (0.2 ml) of a suspension of conidia of G. cingulata (lo6 ml-l) were added to each tube under aseptic conditions and cultures wereincubatedinan orbital shaker (80rmin-1) at 22 “C for 4Oh. Aliquots (0.2 ml) of a solution (2 mg ml-l) of the phytoalexin were added to each tube and the tubes were incubated as described above. Four tubes Corn each treatment were removed at 0, 30, 60 min; 2,4, 6 and 24 h intervals and filtered through Whatman No. 1 paper to remove mycelial mats. The filtrates were extracted in ether and the phytoalexin concentrations estimated as described above. RESULTS

Antifngal activity in immature C. annuum fruit tissueinoculated with G. cingulata Juice expressed from C. annuum fruit tissue 24 h after inoculation with G. CinguZata produced inhibition zones around wells in agar plates seeded with G. cingulata (Fig. 1). Juice expressed from wounded, uninoculated fruits at that time was almost as inhibitory as the juice from inoculated fruits but that from unwounded, uninoculated control fruits was not inhibitory. The toxicity of juice from the wounded, uninoculated tissue increased only slightly during a further 4 days incubation while that from the inoculated fruits increased considerably, especially after 3 days incubation (Fig. 1). Distinct zones of inhibition were produced at RF 0.73 on chromatograms of ether extracts of inoculated fruit tissue oversprayed with C. cladospori&s. The ether extracts

Post-infectionally

formed antifungal

165

compounds

Incubation time (days) FIG. 1. Diameter of inhibition zones produced by juice expressed from immature C. anaaam fruit wound-inoculated with G. cingtdata (0), wounded controls (0) and unwounded tissue (A) on agar seeded with conidia of G. cingduta.

of unwounded control fruits did not cause any inhibition of C. cladosporioah but the extracts from wounded control fruit produced small areas of partial inhibition also at RF O-73. A second set of plates sprayed with G. cingulata developed zones of inhibition which were much larger than those on the plates assayed with C. clados~orio&s. To ensure that this toxic compound, capsicannol [4], was not an artefact of the extraction process, juice expressed from inoculated fruit tissue was chromatographed directly and the inhibitory zone was found to be at the same RF value as with the ether extracts. The concentration of capsicannol which accumulated in wound-inoculated fruit tissue after 18 h incubation, was estimated to be slightly less than O-1 mg g-1 fresh wt and increased to O-8 mg g-l fresh wt after 96 h incubation (Fig. 2). Capsicannol

grm 1

ripening

1 fully ripened

Days after inoculation

FIG. 2. Concentration of capsicannol (a), determined by t.l.c., and capsidiol (H) and capsenone (IJ), determined by g.l.c., in tissue of C. U~WWIIfruit at intervals during ripening after wound-inoculation of green, immature fruit with G. cingulata.

166

N. K. B. Adikaram

et a/.

was also produced in intact fruit tissue inoculated with G. cingulata but in rather lower concentrations, 0.45 mg g-1 fresh wt having accumulated after 96 h incubation (Fig. 2). Capsicannol accumulated in the largest concentrations in tissue immediately below the surface of the fruit; O-95 mg g-l fresh wt accumulated in the first 1 mm tissue. Smaller amounts were present in tissue at greater distances from the surface and at depths greater than 3 mm less than O-2 mg g-r fresh wt was extracted. EDs, value of fw$ed caflsicannol The solubility of purified capsicannol, obtained as previously described [4], was very low in water but was enhanced by the presence ofjuice (1%) expressed from immature fruit in the solution. A solution containing O-5 mg ml-l capsicannol totally inhibited germination of conidia of G. cingulata and the EDs, value was estimated to be 0.35 mg ml-l. The ED,, value for capsidiol assayed with G. cingulata in the same manner was O-03 mg ml-l. Phytoalexin accumulation in immature fruit tissue treated with an elicitor comfionentfrom G. cingulata Preliminary experiments suggested that the elicitor in this host-pathogen combination might be similar to those isolated from the mycelial walls of Colletotrikhum lindemuthianum [S] or C. musae [S] which caused browning and phytoalexin production when applied to surfaces of Phaseolusvulgar& and banana fruit respectively. Mycelial wall extracts from G. cingulata produced brown coloured, depressed lesions of 2 to 4 mm diameter on wounded immature fruits. Unwounded fruits treated with elicitor did not develop any immediate visible lesions; however microscopic examination revealed that some epidermal cells had turned brown. The lesions caused by the elicitor on immature fruits were distinctly larger than those produced by conidia of G. cingulata. Immature fruit tissue treated with elicitor after wounding accumulated larger quantities of capsicannol than tissue wound-inoculated with conidia of G. cingulata; l-3 mg g -1 fresh wt after 4 days incubation [Fig. 3(a)]. Extracts of unwounded fruit tissue treated with elicitor also contained capsicannol but in lower concentrations than elicitor treated wounded tissue. Neither capsidiol nor capsenone were detected in elicitor treated immature fruit tissue. Dialysed mycelial wall extracts (3 ml aliquots) were fractionated on a Sepharose 6B (Pharmacia, Ltd) column equilibrated with O-1 M Tris-HCl buffer @H 6.5) containing 0.5 M KCl. The elicitor was detected in a single peak and was estimated to have a molecular weight of approximately 270 000 daltons. Antifungal a.ctivi& in ripe fruits of C. annuum inoculated with G. cingulata Crude juice expressed from ripening and red, fully ripened fruit tissue taken 3 days after inoculation produced inhibition on agar plates seeded with conidia of G. cingulata in which 2 distinct zones were visible: an inner zone showing complete inhibition and a peripheral zone of partial inhibition. The juice from inoculated fully ripened fruits was more toxic to the fungus than that from ripening fruit (Table 1). Juice from unwounded control tissue was not toxic but wounded fruit tissue taken from fully ripened fruits was slightly toxic.

Post-infectionally

formed antifungal

2 f f ‘O” T, 0.75i? . ) ;MD6 .i 02!5 :: 0

0

167

compounds

1 20

30

40 Dap alftel* inoculation

FIG. 3. (a) The concentration of capsicannol (O), determined by t.l.c., in tissue of C. annuum fruit at intervals during ripening after treatment of immature fruit with elicitor. (b) The concentration of capsidiol ( n ) and capsenone (O), determined by g.l.c., in tissue of ripe C. annuum fruit treated with elicitor. 1

TABLE

Diameter of zones of inhibition and partial inhibition produced by juice expressedfromripening and fdb ripened C. annuum fruit tissue3 a@ after inoculation with G. cingulata OIZagar seeded with conidia of G. cingulata Mean diameter of inhibition Ripening fruit Treatment Inoculated with G. cingulata Wounded control Unwounded control

zones (mm) Fully ripened fruit

1st zone”

2nd zone

1st zone

2nd zone

11.6

25.3 8.0 8.0

17.0

34.4

11.0

16.3

8.0

8.0

s.e. for means = 0.46. a First zone, complete inhibition; * Diameter of wells.

8.0b 8.0

2nd zone, partial inhibition.

Ether extracts of ripening or fully ripened fruit tissue made 3 days after woundinoculating with G. cingulata produced large inhibition zones at RF O-73 on t.1.c. plates developed with solvent A indicating the presence of capsicannol. Zones of inhibition at RF O-38 and at RF O-52 were also visible on the t.1.c. plates corresponding with authentic samples of capsidiol and capsenone respectively. Capsidiol was not observed consistently in all the experiments carried out but was found more often in fruits inoculated at the ripening stage than at the fillly ripened stage. Ether extracts from wounded-uninoculated ripening fruits contained a trace of capsicannol but no capsidiol. Ether extracts of tissue from inoculations made on the intact surface of ripening fruits contained larger quantities of capsidiol but smaller quantities of capsicannol than the extracts of wound-inoculated tissue.

168

N. K. B. Adikaram

et a/.

Phytoalexins in dajksatesfrom fmit cavities Diffusates collected after introducing a suspension of conidia of G. cingulata into green, immature fruits contained less than O-02 mg ml-l capsicannol and neither capsidiol nor capsenone were detected. Diffusates collected from ripening fruits similarly injected with a suspension of conidia of G. cingulata contained relatively large quantities of capsidiol (0.05 mg ml-l) and traces of capsicannol and capsenone. No antifungal activity was detected in diffusates obtained with sterile distilled water from either immature or ripening fruits. Phytoalexins in tissue at various stages of lesion developmentafter inoculation of immature fruits with G. cingulata Maximum quantities of capsicannol accumulated in immature fruits 4 to 5 days after wound-inoculating with G. cingulata (Fig. 2). As the fruit began to ripen, approximately 10 days after inoculation, the quantity of capsicannol had declined considerably and capsidiol had begun to accumulate (Fig. 2). The quantity of capsicannol in the tissue continued to decline to O-2 to O-3 mg g-1 fresh wt as the fruits ripened and remained at that level until the fruits were fully ripened. No capsicannol was detected in rotted tissue. Capsidiol reached maximum concentrations in the later stages of ripening but declined rapidly as fruits became fully ripened. The disappearance of capsidiol coincided with the accumulation of capsenone (Fig. 2). A fourth compound, which produced zones of partial inhibition at RF O-67 on chromatograms developed in solvent A, was detected in extracts of fruits 30 days after inoculation. This compound, like capsenone, increased as the fruit ripened and was apparently present in largest amounts in rotted tissue. Degradation of phytoalexins in elicitor treatedfruits Capsicannol continued to accumulate in fruit tissue after treatment with elicitor at wounded sites, while the fruits remained green and immature, and maximum concentrations of l-25 mg g-l fresh wt were extracted 10 days after treatment [Fig. 3(a)]. As fruit ripening commenced the concentration of capsicannol declined rapidly and continued to decline to less than O-1 mg g-l fresh wt in fully ripened fruits. Capsidiol accumulated in elicitor treated ripening fruit to a maximum concentration of approximately 0.05 mg g-l fresh wt in 3 days but thereafter the concentration declined [Fig. 3(b)]. Capsenone also accumulated in the elicitor treated ripening fruit although more slowly than capsidiol but the concentration continued to increase after 5 days incubation [Fig. 3(b)]. Degradation of capsidiol and capsicannolin vitro by G. cingulata Glomerella cingulata metabolized capsidiol to the less toxic compound capsenone in a similar manner to that described for other fungi [I.?]. The concentration of non-toxic levels of capsidiol in replacement liquid cultures decreased rapidly over a 6 h incubation period and capsenone accumulated at a corresponding rate. Neither capsidiol nor capsenone was detected after 24 h incubation. The metabolism of capsicannol by G. cingulata was slower than that of capsidiol. The concentration in culture filtrates was reduced by approximately 50% during a 24 h incubation period and none was detected after 48 h.

Post-infectionally

formed antifungal

compounds

169

DISCUSSION

Immature C. annuum fruit tissue responded to inoculation with conidia of G. cingulata by accumulating considerable quantities of the fungitoxic compound, capsicannol. Stoessl et al. [II], who investigated the accumulation of post-infectionally formed antifungal compounds in ripening Cap&m fruit, did not demonstrate the presence of capsicannol. Their investigations relied upon the diffusion of potentially fungitoxic compounds into water placed inside the fruit cavity. In similar experiments conducted in this laboratory only a trace of capsicannol was detected in diffusates. It is probable that the very low water solubility of this compound restricted diffusion out of the host tissue. Accumulation of capsicannol took place within 18 h after inoculation and reached a maximum approximately 4 days after inoculation. The high concentration in the superficial tissues at the inoculation sites in immature fruits was sufficient to account for the inhibition of the pathogen. Capsicannol was not detected in healthy Cap&urn fruit tissue but small quantities accumulated as a result of wounding. Capsicannol accumulated rapidly in ripe fruit tissue after inoculation with G. cingulata in quantities apparently sufficient to restrict the development of the pathogen. Necrosis, however, extended further in the ripe fruits hence it is probable that the concentration of capsicannol per lesion volume was lower than in inoculated immature fruit tissue. In progressive lesion tissue, the concentration of phytoalexins had declined to very low levels, insufficient to inhibit the pathogen. The reduction in capsicannol concentration during ripening was accompanied by the accumulation of a compound at RF O-67. The pattern of decline of capsicannol and the increase in the quantity of this compound suggested that the latter may be a degradation product of the former. The nature of the compound at RF O-67 was not investigated but it was apparently much less fungitoxic than capsicannol. Capsicannol concentration declined in elicitor treated tissue as rapidly as in fruits inoculated with the pathogen. Since fungal enzymes would have been destroyed during the preparation of the elicitor host enzymes must have been responsible for this observed decrease in capsicannol concentration. The more fungitoxic compound capsidiol was not detected in immature fruit tissue, in response to infection by the pathogen, indicating that this compound did not contribute to the resistance mechanism of immature fruits. Capsidiol accumulated in ripening fruits in the presence of the pathogen but was rapidly converted to capsenone in fully ripened fruits. Capsidiol/capsenone conversion occurred in fully ripened fruits treated with the elicitor indicating that host enzymes were also involved in this process. Stoessl et al. [13], in fact, suggested that capsidiol might be a normal metabolite of Cap&urn fruits which, in healthy tissue, is metabolized as rapidly as it is formed and that accumulation in infected tissue might represent a block in metabolic utilization rather than stimulated biosynthesis. Capsidiol was metabolized by G. cingulata in vitro first to capsenone which was also metabolized to non-toxic products. Capsidiol was also metabolized in vitro and in vivo by a number of other fungi pathogenic on Capsicumfruits [ 12, 131. In an attempt to correlate in vitro data with the concentrations of capsidiol in Capsicumfruits, Stoessl et al. [I31 concluded that the ability of a fungus to oxidize capsidiol did not appear to be specifically associated with pathogenicity, and they suggested that for Capsicum

170

N. K. B. Adikaram et a/.

fruits under natural conditions other factors were more important than capsidiol accumulation. Results obtained here would suggest that capsidiol accumulation is associated only with ripening &it. It was not detected in arrested lesions produced by either G. cingulda or by Colletotrichum capski or B. cimrea [I]. It was, however, produced in progressive lesions in immature fruit caused by B. cinerea [ij and in progressive lesions initiated by iron-depleted conidia of G . cingzd~tu [3] but localized ripening had occurred at the inoculation sites. The present investigation would suggest that capsicannol is an important factor in the resistance of immature C. annuumfruits but that capsidiol probably plays a r61e in prolonging quiescence in the ripening fruit. We wish to thank Dr A. Stoessl for providing samples of capsidiol and capsenone. One of us (N.K.B.A.) wishes to thank The Queen’s University of Belfast for providing a Visiting Student Support Grant. REFERENCES 1. ADIKARAM, N. K. B. (1981). A study of latent infection of fruit of Ca@nn spp. by Colletottichum cap&i and Glomerellu cingulata. Ph.D. Thesis. Queen’s University of Belfast. 2. ADIRARAM, N. K. B., BROWN,AVERIL E. & !hNBURNE, T. R. (1982a). Observations on the infection of Ca@icumannuum L. fruit by Colletotrichum capsici and Glomerella cingulata. Transactions of the British M~ological Society. (In press.) 3. A~KARAM, N. K. B., BROWN,AVEXIL E. & SWINBURNE,T. R. (10826). Rotting of immature Ca&icumf~tescens L. (C. annuum) fruit by iron depleted Glonterellacingulata (Stonem.). Physiological Plant Pathology 21, 171-177. T. R. 4. ADnL4RAM, N. K. B., GRIMSHAW,J., GRIMSHAW,J. T., BLAKE, P., AUSTIN, D. & +%INBURNE, (1982). Capsicannol, a sesquiterpenoid phytoalexin from immature Cajsicum annuum L. fruit. (In preparation). 5. ANDERSON, A. J. & ALBERSHEIM,P. (1975). Host-pathogen interactions. VII. Isolation of a pathogen synthesized fraction rich in glucan, that elicits a defence response in the pathogens host. Plant Physiology56,286-291. T. R. (1980). The resistance of immature banana fruits to anthrac6. BROWN,A. E. & SWXNBURNE, nose [Colletotrichum musae (Berk. & Curt.) AI-X.]. Phytopathologische,@tsch@? 99, 70-80. 7. Joru~s, D. R., U~wnu, K. H. & WARD, E. W. B. (1975a). The significance of capsidiol induction in pepper fruit during an incompatible interaction with Phytophthora infestans. Phytopathology 65, 1286-1288. 8. JONES,D. R., UNWIN, K. H. & WARD, E. W. B. (19756). Capsidiol indiction in pepper fruit during interactions with Phytophthora cap&i and Monilia fnccticola. Pfiytopatholo~ 65, 1417-142 1. 9. KLARMAN, W. L. & STANFORD, J. B. (1968). Isolation and p&cation of an antifungal principle from infected soy beans. Lift Scimces7, 1095-l 103. 10. MUIRHEAD, I. F. & DEVERALL,B. J. (1981). Role of appressoria in latent infection of banana fruits by Collctotrichum musae. Physiological Plant Pathology 19, 77-84. 11. STOESSL, A., Um, C. H. & WARD, E. W. B. (1972). Post-infectional inhibitors from plants. I. Capsidiol, an antifungal compound from Capsicumftiscens. Phytopathologzkhe ,@Mri~t 74, 141-152. 12. STOESSL, A., UNWIN, C. H. & WARD, E. W. B. (1973). Post-infectional inhibitors from plants: Fungal oxidation of capsidiol in pepper fruit. Phytupathology63, 1225-1231. 13. STOESSL, A., ROBINSON, J. R., ROCK, G. L. & WARD, E. W. B. (1977). Metabolism of capsidiol in sweetpepper tissue: somepossibleimplicationsforphytoalexinstudies. Phytopathology67 (I), 64-66. T. R. (1976). Stimulants of germination and appressoria formation by Co&tot&urn 14. SWINBURNE, musae (Berk. & Curt.) Arx. in banana leachate. Phytopathobgische