Detection of rishitin in tomato fruits after infection with Botrytis cinerea

Physiological Plant Pathology (1981) 19,243-248

Detection of rishitin in tomato fruits after infection with

Botrytis cinerea ]. A.

GLAZENER

and C. H.

WOUTERS

Phytopathological Laboratory 'Willie Commelin Scholten', Javalaan 20, 3742 CP Baarn, The Netherlands (Accepted for publication June 1981)

The rille of the phytoalexin rishitin was studied in young tomato fruits in relation to infection by Batrytis cinerea. After wound inoculation, carried out by removing 1 mm of the outer epidermal layers and using a conidial suspension of B. cinerea as inoculum, rishitin was found in the infected tissue. In vitro experiments showed that rishitin at a concentration of 10 J.lg ml- 1 had considerable antifungal activity towards B. cinerea, retarding mycelial growth on solid media from agar discs, and almost completely inhibiting growth of l-day-old sporelings in liquid cultures. However, no rishitin was detected in lesions developing from surface inoculations in which spores were dusted over the fruit. In such cases defense mechanisms, other than rishitin accumulation, must be involved. These may include the pre-formed tomatine and post infectional Iignification.

INTRODUCTION

Young tomato fruits develop small necrotic lesions after infection with Batrytis cinerea [1, 13]. In these necrotic areas fungal hyphae are surrounded by both dead and living cells which contain polyphenolic deposits in their walls [9]. The polyphenols could well be lignin, formed by the host tissue as a barrier against B. cinerea [12]. Although precursors oflignin were found to accumulate within 24 h of inoculation, necrosis only becomes visible after 36 h and lignin could only be demonstrated clearly after 72 h [5]. Thus other mechanisms are probably involved during the early stages of infection in preventing fungal spread beyond the first few cell layers. Young tomato fruits contain the glycoalkaloid tomatine with the highest concentrations in the skin of the fruit (14]. B. cinerea was found to be sensitive to tomatine in vitro at concentrations found in vivo. Tomatine could therefore be responsible for the limited growth of B. cinerea in young tomato fruits. Studies on other host-pathogen combinations in the Solanaceae [11] have shown that phytoalexins may playa role in disease resistance. Tomato fruits produce the sesquiterpene rishitin, after wound inoculation with Pbytophthora infestans [10] and Cladosporium fuluum [2]. C.fulvum also induced 4 other fungitoxic compounds, 2 of these appearing to be polyacetylenes [3]. In view of these results the question arises as to whether phytoalexins could play a determinative role in the interactions between tomato fruit and B. cinerea and the present study reports an investigation of the role of rishitin in this host-pathogen combination. 0048-4059/81/050243+06 S02.00/0

© 1981 Academic Press Inc. (London) Limited

244

J. A. Glazener and C. H. Wouters

MATERIALS AND METHODS

Fungal material B. cinerea was isolated from naturally infected tomato plants and maintained on PDA. Conidia were used for all inoculations, either obtained from cultures grown on PDA plates for 10 to 14 days or from 4-week-old cultures grown on autoclaved tomato leaves. All cultures, except where indicated, were incubated at about 20°C under continuous light. Cladosporium herbarum was used for bio-assays on thin layer chromatography plates. This fungus was maintained on PDA at approx. 20°C under continuous light. Conidia for the bio-assays were harvested from PDA plates seeded with conidia and incubated in darkness at 19 °0 for 7 days. Isolation of rishitin Rishitin was isolated from potatoes, which had been inoculated with Erwinia carotovora var, atroseptica according to the procedure described by Lyon [6]. Purity of the rishitin was checked by thin layer chromatography and by gas chromatography. Plant material Tomato plants (Lycopersicum esculentum Mill.) cultivar "Moneymaker" were grownina glasshouse at 22 to 26°C, under an approximately 16 h day. Green fruits with a diameter of 2 to 3 em were harvested and inoculated in 2 ways, either surface inoculated or wound inoculated. In the first case conidia were dusted over fruits by holding a fungal culture over them and gently rubbing off the conidia with a brush. Wound inoculations were carried out by making a circular incision into the fruit surface using a sterile corkborer, (0'5 em diameter) and removing the enclosed outer skin tissue to a depth of about I mm. One drop of conidial suspension (3'5 X 10 4 spores ml- 1 ) was then placed on the wounded surface. All inoculated fruits were incubated in a plastic box, to maintain 100% r.h., at approximatey 19°C under a 16 h day. After 12 h, the lids of the boxes were removed and the fruits were harvested after incubation for the required period. Uninoculated control fruits were incubated under similar conditions. Sterile water was used to fill the wounds on the wounded controls. Rishitin extraction Plant material from the surface inoculated fruits was obtained by collecting the necrotic areas. Similar parts were taken from the uninoculated controls. The soft infected tissues and the adjacent healthy tissue were harvested from the wound inoculated fruits. This plant material was extracted as described by Price et al. [8] and by Elgersma [4]. Each sample contained 10 to 20 g of tissue. Bio-assays on t.l.c, plates Conidial suspensions of C. herbarum were made by collecting conidia from potato dextrose ager (PDA) plates and suspending them in Czapek-Dox medium. The suspension was sprayed evenly over the developed silica gel plates and the plates were incubated in darkness at 22°C in a moist atmosphere for 4 to 5 days,

245

Detection of rishitin in tomato fruits

Sensitivity ofB. cinerea to rishitin in vitro Rishitin was dissolved in ethanol in various concentrations and added to nutrient media in a final concentration of 0·5 % ethanol. Mycelial growth was measured on PDA agar to which various concentrations of rishitin had been added. When the plates (9 em diameter, with 12 ml agar) had solidified, a 4 mm agar disc of a 3 to 4-day-old culture of B. cinerea was placed in the centre and they were incubated at 21 °0 in the dark. Radial growth was measured twice a day for several days after inoculation. Mycelial production from conidia was determined in a liquid medium containing per litre of distilled water: 10 g KNOll, 5 g KH 2 P0 4 , 0·25 mg MgS0 4.7H 20, a trace of FeOla and 1% glucose; 5 X lOa spores ml- 1 medium were used as inoculum. After an incubation period of24 h on a shaker at 20 °0, rishitin was added in different concentrations. After another 3 days of incubation the mycelium was collected on tared filter paper and dried at 80 °0 for 24 h, after which the dry weight was determined. Metabolism of rishitin The method described by Lyon [7] was used. Conidial suspensions were inoculated into the mineral salt solution as described above. After 4 days incubation, 180 Ilg rishitin was added to each flask, giving a final concentration of 7·2 Ilg rril- 1 medium. The incubation was stopped by adding 50 ml ethanol to the flasks at 0,2,7 and 24 h after the addition of the rishitin, Rishitin was extracted and the amount determined quantitatively using a gas chromatograph. RESULTS

The two types of inoculation gave different results. Surface inoculation led to the development of necrotic spots after .36 h and in some cases blisters were found [13]. Wound inoculations resulted in a fast-spreading soft rot lesion, without browning, which increased in diameter by about 0·3 em day-I. Table I shows the amount of rishitin detected in the infected tissues. Lesions in surface inoculated fruits reached their maximum size 48 h after inoculation, and the last sample was taken 24 h after expansion had ceased. The wound inoculated fruits were first sampled, 90 h after inoculation, when large quantities of tissue from TABLE 1 Rishitinproduction (Jigg -l.fresh wt) byyoungtomatofruit tissue, ofur woundandsurface inoculation with Botrytis cinerea

Hours after inoculation

24 48

72 90 94 116

Surface inoculations Inoculated Control

o o o

Wound inoculations Inoculated Control

o o o 0·2

o

1-4

°o

0·7

Data are the average of at least 3 replicates. 0, rishitin not detectable; -, no determinations were carried out (see text).

246

J. A. Glazener and C. H. Wouters

linearly expanding lesions could be obtained. Rishitin was only detected in the samples obtained from the wound inoculated fruits. The t.l.c. bio-assays of extracts from the surface inoculated fruits showed no fungitoxic spots. Extracts from wound inoculated tissue did produce fungitoxic compounds including rishitin. After 90, 94 and 116 h incubation, 3 spots were seen on t.I.c, plates where fungal growth was inhibited. Using cyclohexane : ethylacetate (1 : 1, vfv) as the developing solvent the R F values were, 0·27 to 0,29, 0·55 to 0'59 and 0·72 to 0·76. These spots were not found in healthy tissue extracts, although occasionally a very faint spot was found at R F 0·72. The spot at R F 0·27 to 0·29 turned red when H 2S04 was added, as did the rishitin which was included as a reference. The identities of the other two spots were not determined. Sensitivity of the strain of B. cinerea to rishitin was tested on an agar medium (Fig. 1) and in a liquid medium (Table 2). On agar containing the highest amount of rishitin, 30 ug ml "", the mycelium of the inoculum disc initially grew extensively on

30r------------------------,

~A

C

/'8

0

E 20

E

••~;;;;;;;;;;;;;;,,!-L>.----o

40

10

90

60

50

Tlrne Ih )

FIG. 1. Influence of increasing amounts of rishitin in agar on growth of Botrytiscinerea. O~lg ml- i ; . , 51lg ml- 1 ; 0, 1OIlgm1- 1 ; are averages from 5 replicates.

0,

. ,

TABLlI

20 Ilgml- i ;

s,

30Ilgml-1. Data points

2

Sensitivity of germinating conidia ofBotrytis cinerea to varying concentrations of rishitin. in liquid media Concentration of rishitin (Ilg ml- i )

o 5 10 15

20

Mycelial dry wt in mg 10 ml " ! medium 90·8 39-6 0·9 0·8 0·8

(100%) ( 47%) ( 1%) ( 1%) ( 1%)

Detection of rishitin in tomato fruits

247

the disc itself but there was a lag before hyphae grew out onto the rishitin agar, and the final growth rate was much lower (0,42 mm h -1) than on the control medium

(0'71 nun h- 1) . B. cinerea responds more sensitively to rishitin when growing in a liquid medium as can be seen in Table 2. There was a marked reduction in mycelial growth at concentrations of between 5 and 10 Ilg ml- 1 of medium. Also, feeding low levels of rishitin to actively growing cultures in a liquid medium to determine whether B. cinerea was able to metabolize rishitin gave results similar to those of Lyon [7]. After 24 h of incubation, less than 25% of the added rishitin could be recovered.

DISCUSSION

No rishitin was detected in the lesions formed in surface inoculated tomato fruits, indicating that either the cells do not produce rishitin when inoculated in this way or that the fungus metabolizes the rishitin immediately upon its formation. In the wound inoculated fruits the outer epidermal layers were removed, thereby removing the tomatine which is concentrated therein [14J, and since the fungus developed to form a sporulating lesion, there is no indication of the presence of other inhibitory factors in significant concentration in the inner tissues. Although rishitin was found in the infected area, the concentrations were rather low. Mycelial growth of B. cinerea is clearly inhibited by rishitin, with germinating conidia being the more sensitive growth phase. However, we have confirmed Lyons [7] finding that B. cinerea can metabolize it, at least when it is present in low concentrations. In the wound inoculated fruits rishitin or the 2 unknown compounds might play a role in slowing down fungal growth, but they can play little if any role in the resistance of whole fruits, since these compounds do not accumulate at any stage. Other workers have also shown that rishitin may be unimportant in resistance mechanisms of tomato to other pathogens. Thus rishitin does not appear to play an important role in resistance to Verticillium albo-airum [4], and it was not found to accumulate in tomato leaves after inoculation with either incompatible or compatible races of C.juluum. However, it did accumulate in fruits in response to both compatible and incompatible races, but in both cases it accumulated to the same amount [2]. The wound-inoculated fruits in our experiments accumulated rishitin to comparable levels to those found by De Wit & Flach [2], although the spore concentrations used in our experiments were lower. They also reported 4 fungitoxic compounds besides rishitin and 2 of these might beidentical to the 2 unidentified fungitoxic compounds found in these studies. It seems likely, therefore, that in surface inoculated young tomato fruits it is not rishitin but the preformed tomatine [14] or the post infectional lignification of cell walls [5] that plays the major role in warding-off the fungus.

Thanks are expressed to Dr K. Verhoeff and Dr D. M. Elgersma for helpful discussions during our studies. The Netherlands Organisation for the Advancement of Pure Research supported this study by a grant to the senior author.

2.48

J. A. Glazener and C. H. Wouters

REFERENCES 1. AINSWORTH, G. C., OYLER, E. & READ, W. H. (1938). Observations on the spotting of tomato fruits by Botryiis cinerea. Annals of AppliedBiology 25, 308-320. 2. DE WIT, P.]. G. M. & FLACH, W. (1979). D ifferential accumulation ofphytoalexins in tomato leaves but not in fruits after inoculation with virulent and avirulent races ofCladosporiumJulvum.

Physiological Plant Pathology 15,257-267. 3. DE WIT, P. ]. G. M. & KODDE, E. (1981). Induction of polyacetylenic phytoalexins in Lycopersicum esculentum after infection with Cladosporium fuloum. Physiological Plont Pathology 18, 143148. 4. ELGERSMA, D. M . (1980). A ccumulation of rishitin in susceptible a nd resistant tomato plants after inoculation with Verticillium albo-atrum. Physiological Plant Pathology 16, 149-15 3. 5. GLAZENER, J. A. (1980). Defense mechanisms in tomato fruit after infection with Botrytis cinerea. Thesis, State University, Utrecht , The Netherlands. 6. LYON, G. D. (1972). O ccurrence of rishitin and phytuberin in po ta to tu bers in ocula ted wi th Erwinia carotouora var. atroseptica. , Physiological Plant Pathology 2, 411-416 . 7. LYON, G. D. (1976). Metabolism of the phytoalexin rishitin by Botrytis spp. Journal of General Microbiology 96, 225-226. 8. PRICE, K. R., HOWARD, B. & COXON, D. T. (1976). Stress metabolite production in potato tubers infected by Phytophthora irfestans, Fusarium avenaceum and Phomaexigua. Physiological Plant Pathology 9, 189-197. 9. RIJKENIIERG,·F. H. ]., DE LEEuw, G. T. N. & VERHOEFF, K. (1980). Light and electron microscopical studies on the infection of tomato fruits by Botrytis cinerea. Canadian Journal of Botany 58, 1394-1404. 10. SATO, N. , TOMIYAMA, K ., KATSUI, N. & MAsANUNE, T . (1968). Isol ation ofrishitin from tomato plants. Annals of the Phytopathological Society ofJapan 34, 344-345. II. STOESSL, A., STOTHERS, J. B. & WAIUJ, E. W . II. (1976). Sesquiterpenoid stress compounds of the Solanaceae. Phytochemistry 15,855-872. 12. VAN MAARSCHALKERWEERD, M. & VERHOEFF, K . (1976). Lignification as a poss ible defense mechanism in tomato fru its after infection by Botrytis cinerea. Acta Botanica Neerlandica 25, 256 (Abstr.), . 13. VERHOEFF, K. (1970). Spotting of tomato fruits caused by Botrytis cinerea. Netherlands Journal oj Plant Pathology 76, 219-226. 14. VERHOEPF, K. & LIEM, J. 1. (1975). Toxicity of tomatin to Botrytis cinerea in relation to latency. Phytopathologische Z eitschrift 82, 333-338.