Planta
Planta 135, 301-306 (1977)
9 by Springer-Verlag 1977
Ethylene Evolution by Rust-infected, Detached Bean (Phaseolus vulgaris L.) Leaves Susceptible and Hypersensitive to Uromyces phaseoli (Pers.) Wint. * P. Montalbini** and E.F. Elstner*** Institut ffir Botanik und Mikrobiologie, Technische Universitfit Mtinchen. Arcisstr. 21, D-8000 Mfinchen, Federal Republic of Germany
Abstract. Ethylene production in leaves of susceptible and hypersensitive varieties of beans has been followed after inoculation with Uromyces phaseoli. Four different states of ethylene evolution are distinguishable: (1) 13 h after inoculation and concomitant to the penetration of the fungal mycelium through the stomata, all varieties show an outburst of ethylene with significant differences between the three varieties. (2) After 36 h postinoculation, in all three varieties ethylene evolution is scarcely higher than in noninfected leaves. (3) Starting 59 h after inoculation, only in the hypersensitive variety 765 (which shows the lowest ethylene production after 13 h), a second, very strong ethylene outburst is observed. (4) From 125 h after inoculation, significant ethylene production is not observed in any variety. At this time, characteristic symptoms are expressed in susceptible leaves (differentiation of uredosori) and in the hypersensitive variety 765 (large brown necrotic spots); no macroscopic symptoms are observed in the hypersensitive variety 814, which exhibits the strongest ethylene outburst 13 h after inoculation. The capacity for ethylene formation after mechanical wounding (" point freezing ") is almost identical in healthy leaves of all three varieties. This capacity is still preserved after the first ethylene outburst 36 h after infection.
and Goeschl, 1969; Abeles, 1973), released in very small amounts under physiologic conditions. During fruit ripening, flower fading, abscission, and senescence of leaves, an increased evolution of this gas is frequently observed (Abeles, 1973). An outburst of ethylene is a known phenomenon linked to mechanical and chemical injury of plants. It is also observed during the interaction between host and pathogen after infections, particularly in those complexes leading to hypersensitivity (Williamson, 1950; Ross and Williamson, 1951 ; Freebairn and Buddenhagen, 1964; Smith etal., 1964; Stahman et al., 1966; Chalutz and Devay, 1969; Balazs et al., 1969; Nakagaki et al., 1970) and seems to be associated with the necrotic process. Wheat leaves infected with Puccinia graminis tritici, or tomato plants infected with Verticillium albo atrum or with Fusarium, however, behave in the opposite way (Daly et al., 1970; Pegg and Cronshaw, 1976; Gentile and Matta, 1975) : they produce higher amounts of ethylene during the expression of susceptibility. In this communication we present data concerning ethylene evolution during the incubation cycle of Uromyces phaseoli in bean leaves susceptible and hypersensitive to the parasite in comparison to the effect of mechanical wounding (" point freezing ").
Key words: Ethylene - Hypersensitivity - Phaseolus - Rust infection - Uromyces. Materials and Methods Introduction Ethylene is known to be an endogenous regulator of growth and development in higher plants (Pratt * This work was supported by the Deutsche Forschungsgemeinschaft, by the Kleinwanzlebener Saatzucht AG (Einbeck, FRG) and by a NATO fellowship to P.M. ** Permanent address: Istituto di Patologia Vegetale, Universita Perugia, Italy *** To whom correspondance should be addressed
Bean plants (Phaseolus vulgaris L.), susceptible (var. Pinto 111) and hypersensitive (var. 814 and var. 765) to rust infection (Uromyces phaseoli), were grown in the greenhouse at 18 22~ under natural light conditions supplemented with artificial illumination (11 h photoperiod). The two primary leaves were inoculated by spraying with a suspension of uredospores, yielding saturating infection. At different times after inoculation, healthy (Fig. 1a) and infected leaf blades were carefully detached and arranged inside of plastic containers (10 cm in diameter, with ca. 20 ml volume) with rubber-sealed outlets for gas analysis. After 1, 2 and 3 h of incubation in the dark, 1 ml of the gas was analyzed as described (EIstner and Konze 1976). Ethylene concentrations were calculated
302
P. Montalbini and E.F. Elstner: Ethylene Evolution by Rust-infected Leaves
Fig. l a . Healthy leaf from Phaseolus vulgaris var. Pinto 111. b. Infected leaf from Phaseolus vulgaris var. Pinto 111, ca. 9 days after inoculation with Uromyces phaseoli. Differentiation of uredosori is visible, e. Infected leaf of Phaseolus vulgaris var. 765, ca. 9 days after inoculation with Uromyces phaseoli. Large brown lesions (but no differentiation of uredosori) are visible, d. Infected leaf of Phaseolus vulgaris var. 814, ca. 9 days after inoculation with Uromyces phaseoli. No macroscopically visible symptoms
by comparison with dilutions of authentic gas samples. Point freezing of bean leaves was performed as described (Elstner and Konze, 1976). For each 100 mg of leaf weight, 4 frozen areas were set in approximately regular distances of 1 cm, by touching a stainless steel rod with a diameter of 3 m m (kept at liquid nitrogen temperature) for 2 s on the leaf surface.
Characteristics of the Infection Within the Varieties Under the preceding conditions, flecks were very evident on Pinto 111 around 125 h after inoculation, but were barely visible 7 0 h after inoculation. Differentiation of uredosori started ca. 8-9 days after inoculation (Fig. 1 b). In variety 765, the hypersensitivity started to be visible ca. 60 h after inoculation as very small depressions of the epidermis layer, a first macroscopically visible s y m p t o m of the histologic collapse. In the subsequent 20 30 h the p h e n o m e n o n became more pronounced and ca. 100 h after the inoculation the lesions became brown coloured, indicating the end of the hypersensitivity process (Fig. 1 c). In variety 814 no symptoms of the disease are macroscopically visible. F r o m microscopic observations (Marte and Montalbini, 1972) it is known that hypersensitivity is limited to very small histologic areas. Fluorescence of the infected cells, to which collapase is closely related, appears around 2 0 h after inoculation (Fig. 1 d).
Results
I. Ethylene Production as a Response after Infection Suspensions of fungal spores as used for the infection did not produce any ethylene when kept in closed vessels for several days and there was no detectable ethylene production during spore germination on the leaves during the first 8 h after inoculation. Approximately 10 h after inoculation with a spore suspension of Uromyces phaseoli (yielding a very strong, "saturating" infection), in all three varieties (Pinto 111 = susceptible; 765 and 814=hypersensitive), an outburst of ethylene could be observed (Fig. 2). This first outburst (state 1) corresponds in timing to the production of substomatal vesicles in the stomatal cavities (cf. Mendgen and Heitefuss, 1975). In the hypersensitive variety 814 significantly more ethylene is produced than in the susceptible variety Pinto 111 or in the hypersensitive variety 765. Approximately 20 h after inoculation, this first ethylene outburst decreases and only negligible eth-
P. Montalbini and ILF. Elstner: Ethylene Evolution by Rust-infected Leaves
303
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Fig. 2. Differences in ethylene production by detached bean leaves from three varieties during the first 36 h after inoculation with Uromyces phaseoli. For experimental details see Methods. e - - e Pinto I l l (susceptible); , - - - A var. 814 (hypersensitive) ; x --- x var. 765 (hypersensitive). The points represent means (with standard error) of 12 values (4 probes from 3 independent experiments)
36
Time [hi
ylene formation is observed in all three varieties 36 h after inoculation (state 2). At 20 h after inoculation, the first haustoria of the fungal mycelium are produced in the mesophyll of the leaves (compare Fig. 3 a and 3 b).
a I0-14h after inoculation
Substornato[
~
b
ca. 20h after inoculation
Approximately 60 h after inoculation, a very strong ethylene outburst can be measured in the hypersensitive variety 765, lasting for ca. 50-60 h. This variety exhibits the lowest ethylene response during the formation of the substomatal vesicle (state 1).
c
ca, 60h after inoculation
vesicle
multiple houstoria
first haustoria
ppressorium
2s
state 1
state
2
state 3
Fig. 3. Schematic drawings of three states of fungal development on and in bean leaves (from Sempio and Caporali, 1958, and after Mendgen and Heitefuss0 I975)
304
P. Montalbini and E,F. Elstner: Ethylene Evolution by Rust-infected Leaves
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Time [hi Fig. 4. Ethylene formation during the infection cycle of U r o m y c e s phaseoli in detached leaves of susceptible and hypersensitive varieties vulgaris. For experimental details see Methods. In the controls (healthy=noninfected leaves) we observed an average of P h a s e o l u s ethylene formation of below 0.5 nmol/g fresh weight x h. o - - o Pinto 111 (susceptible); A. 9 9 var. 814 (hypersensitive); x - - - x vat. 765 (hypersensitive). The points represent means (with standard error) of 12 values (4 probes from 3 independent experiments)
In the varieties Pinto 111 (susceptible) and 814 (hypersensitive) no second outburst of ethylene during this state (state 3) is observed (Fig. 4), although in the susceptible variety Pinto 111 at this time a strong mycelium is spreading throughout the mesophyll (cf. Fig. 3 c).
H. Ethylene Production as a Response after Mechanical Wounding
The ethylene/ethane ratio in the gas phase of incubated leaf disks or leaves after wounding by point freezing seems to be a good indicator for the integrity of green leaves, as far as the intactness of the compartmentalization inside the cells is concerned (Elstner and Konze, 1976). Therefore, the effect of point freezing on ethylene and ethane production by healthy and infected (states 2 and 4) bean leaves of the three varieties Pinto 111 (susceptible) and 765 and 814 (Hypersensitive) has been studied.
As shown in Table 1, all three varieties show similar responses after point freezing of parts of the leaf surface (cf. Methods), namely an approximately 7to 10-fold increase of ethylene production accompanied by ethane formation. Table 1. Effect of wounding (point freezing) on ethylene- and ethane formation by noninfected bean leaves of three varieties Variety
Untreated
After point freezing"
ethylene
ethane
ethylene
ethane
Pinto 111 (susceptible)
3.12_+3.3
0
27.52_+14.21
5.8+5.89
Var. 814 (hypersensitive)
3.12+2.8
0
32.22_+18.5
4.7_+3.08
Var. 765 (hypersensitive)
4.32+_4.9
0
31.45-+21.23
4.97-+3.52
The numbers ( x 10-11 mol/g fresh weight • h) represent means of 17 independent experiments with standard deviations (-+)
P. Montalbini and E.F. Elstner: Ethylene Evolution by Rustqnfected Leaves
305
Table 2. Effect of wounding (point freezing) on ethylene- and ethane formation by infected bean leaves of three varieties during two different states ~ of fungal development Variety
State 2
State 4
untreated
Pinto 111 (susceptible) Var. 814 (hypersensitive) Var. 765 (hypersensitive)
wounded
untreated
C2H4
C2H6
C2H4
C2H6
6.35_+0.9 5.3_+3.7 5.2_+5.3
0 0 0
41.5_+ 6.6 5.8_+ 3.6 62.5_+10.3 5.0_+ 4.7 7 9 . 0 _ + 3 3 . 8 10.3_+ 5.6
wounded
C2H4
C2H 6
C2H 4
C2H6
30.0_+22.6 20.6_+18.1 9.4_+ 9.5
0 0 0
24.3+_18.9 41.0_+16.] 75.5_+34.5
0 4.0_+4.4 9.7_+9.2
The numbers ( x 10 11 mol/g fresh weight x h) represent means of 5 independent experiments with standard deviations (_+) a The states of fungal (Uromyces phaseoli) development are explained in the text and in Figures 2 and 3
With the applied wounding technique (4 frozen leaf areas, each 3 mm in diameter/100 mg leaf weight), ethane formation on a molar basis represents ca. 10-20% of the observed ethylene formation. All three varieties also show good (although slightly different) response after wounding during state 2 (ca. 36 h after inoculation). During state 4 (ca. 125 h after inoculation) both hypersensitive varieties show a significant ethylene- and ethane response after point freezing. In the susceptible variety (Pinto 111), however, this response seems to be lost, since no increase of ethylene formation or induction of ethane formation is observed (Table 2).
Discussion
The experiments described in this communication were performed in order to study whether the plant hormone ethylene is involved in the expression of susceptibility (cf. Daly et al., 1970; Pegg and Cronshaw, 1976) or of hypersensitivity (cf. Ross and Williams, 1951; Smith et al., 1964; Balazs et al., 1969; Nakagaki et al., 1970) of bean leaves infected by Uromyces phaseoli. As shown in Figures 2 and 3, all three tested varieties (Pinto 11 l = susceptible; var. 765 and var. 814= different degrees of hypersensitivity) exhibit a pronounced ethylene outburst ca. 10 to 14 h after inoculation. This ethylene production corresponds in timing to the fungal penetration through the stomata and the formation of substomatal vesicles. In variety 814 where hypersensitivity is expressed very early (microscopic fluorescence of collapsed cells is visible ca. 18-20 h after inoculation, Marte and Montalbini, 1972), the highest ethylene outburst at this state of fungal development (state 1) is observed. The variety 765, also hypersensitive, but with very late expression of the hypersensitivity symptoms, shows only little ethylene formation at state 1, namely
ca. 25% of the response of var. 814 and ca. 50% of the response of the susceptible variety Pinto 111. There is no significantly (background of healthy leaves!) increased ethylene formation in any of the tested varieties 36 h after inoculation (state 2). In the highly resistant variety 814 and in the susceptible variety Pinto l 11 no further periods of ethylene outburst could be detected during the development of the fungal cycle. Approximately 60 h after inoculation, the variety 765 exhibits a second, but this time very strong ethylene outburst, lasting for ca. 60-65 h (state 3). After 125 h postinoculation, no further ethylene evolution significantly above background could be detected in variety 765. In order to study whether fungal penetration and development in the mesophyll of the leaves is similar to the effects observed after mechanical damage (at least as far as the ethylene response is concerned!), wounding experiments were performed during the various "nonethylene producing" states (healthy, state 2=36 h after inoculation; state 4= from 125 h after inoculation). As shown in Tables 1 and 2, all three varieties show an increased ethylene production after "point fieezing" (as a reproducible wounding technique, cf. Elstner and Konze, 1976) in healthy and infected leaves at state 2 of fungal development. There is one pronounced difference between the fungal influence and the mechanical effect on the leaves, however: after fungal infection, ethane is not detectable in any state as an accompanying gas of ethylene, while after wounding, ethane formation seems to be a characteristic feature of mechanical "decompartmentalization" of living cells (Elstner and Konze, 1976). The ability for increased ethylene production after wounding is preserved (although to various extents) in all three varieties after the ethylene outburst in state 1. In state 4, the ability for increased ethylene formation after wounding seems to be lost only in the susceptible variety Pinto 111, where very strong symptoms of infection (flecking and uredosori formation) were visible at this state.
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P. Montalbini and E.F. Elstner: Ethylene Evolution by Rust-infected Leaves
We conclude from these data that stimulation of ethylene formation after mechanical wounding and after fungal infection constitutes different processes. Since all three varieties respond to the penetration of the fungal mycelium through the stomata, but only var. 765 shows a second ethylene response due to the invasion of the mycelium in the mesophyll (state 3), we conclude that the ethylene response in state 1 and that in state 3 of vat. 765 are also different processes. The reason why there is no second ethylene outburst in var. 814 may simply be the fact that the fungus has been "killed" by the early hypersensitive reaction in state 1. The lack of ethylene response in state 3 of the susceptible variety Pinto 111 is probably not due to an exhausted (by ethylene formation in state 1) ethylene-precursor pool, since wounding of state 2 Pinto 111 leaves shows the same ethylene response as wounding of non-infected leaves. Thus either the precursors for ethylene formation during states 1 and 3 in var. 765 are different from the precursors of ethylene after wounding, or the processes of triggering the conversion of these compounds into ethylene are not the same.
References Abeles, F.B. : Ethylene in plant biology, London-New York: Academic Press 1973 Balfizs, E., G~tborjfinyi, R., Toth, A., Kirfily, Z. : Ethylene production in xanthi tobacco after systemic and local virus infections. Acta Phytopathol. Acad. Sci. Hung. 4, 355-358 (1969) Chalutz, E., De Vay, J.E. : Production of ethylene in vitro and in vivo by Ceratocystisfimbriata in relation to disease development. Phytopathology 59, 750-755 (1969)
Daly, J.M., Seevers, P.M., Ludden, P.: Studies on wheat stem rust resistance controlled at the Sr6 locusl. III. Ethylene and disease reaction. Phytopathology 60, 1648-1652 (1970) Elstner, E.F., Konze, J.R.: Effect of point freezing on ethylene and ethane production by sugar beet leaf disks. Nature 263, 351-352 (1976) Freebairn, H.T., Buddenhagen, I.W. : Ethylene production by Pseudomonas solanacearum. Nature 202, 313 314 (1964) Gentile, I.A., Matta, A. : Production of and some effects of ethylene in relation to Fusarium wilt of tomato. Physiol. Plant Pathol. 5, 27 37 (1975) Marte, M., Montalbini, P. : Microfluorescenza in foglie de fagiolo suscettibile e resistente alla ruggine. Phytopathol. Z. 75, 59-73 (1972) Mendgen, K., Heitefuss, R,: Micro-autoradiographic studies on host-parasite interactions. I: The infection of Phaseolus vulgaris with tritium labeled uredospores of Uromyces phaseoli. Arch. Microbiol. 105, 193-199 (1975) Nakagaki, Y., Hirai, T., Stahmann, M.A.: Ethylene production by detached leaves infected with tobacco mosaic virus. Virology 40, 1-9 (1970) Pegg, G.F., Cronshaw, D.K. : Ethylene production in tomato plants infected with Verticillium albo-atrum. Physiol. Plant Pathol. 8, 279-295 (1976) Pratt, H.K., Goeschl, J.D.: Physiological roles of ethylene in plants. Ann. Rev. Plant Physiol. 20, 541-584 (1969) Ross, A.F., Williamson, C.E. : Physiological active emanation from virus-infected plants. Phytopathology 41, 431438 (1951) Sempio, C., Caporali, L. : L'Uromyees Appendiculatus sul fagiolo e su altre specie: Virulenza e specializzazione. Ann. Fac. Univ. Perugia XlII, 233-277 (1958) Smith, W.H., Meigh, D.F., Parker, J.C.: Effect of damage and fungal infection on the production of ethylene by carnation. Nature 204, 92 (1964) Stahmann, M.A., Clare, B.G., Woodbury, W.: Increased disease resistance and enzyme activity induced by ethylene and ethylene production by black rot infected sweet potato tissue. Plant Physiol. 41, 1505-1512 (1966) Williamson, C.E. : Ethylene, a metabolic product of diseased or injured plants. Phytopathology 40, 205-208. (1950)
Received 3 March; accepted 1 April 1977
Planta 135, 301-306 (1977)
9 by Springer-Verlag 1977
Ethylene Evolution by Rust-infected, Detached Bean (Phaseolus vulgaris L.) Leaves Susceptible and Hypersensitive to Uromyces phaseoli (Pers.) Wint. * P. Montalbini** and E.F. Elstner*** Institut ffir Botanik und Mikrobiologie, Technische Universitfit Mtinchen. Arcisstr. 21, D-8000 Mfinchen, Federal Republic of Germany
Abstract. Ethylene production in leaves of susceptible and hypersensitive varieties of beans has been followed after inoculation with Uromyces phaseoli. Four different states of ethylene evolution are distinguishable: (1) 13 h after inoculation and concomitant to the penetration of the fungal mycelium through the stomata, all varieties show an outburst of ethylene with significant differences between the three varieties. (2) After 36 h postinoculation, in all three varieties ethylene evolution is scarcely higher than in noninfected leaves. (3) Starting 59 h after inoculation, only in the hypersensitive variety 765 (which shows the lowest ethylene production after 13 h), a second, very strong ethylene outburst is observed. (4) From 125 h after inoculation, significant ethylene production is not observed in any variety. At this time, characteristic symptoms are expressed in susceptible leaves (differentiation of uredosori) and in the hypersensitive variety 765 (large brown necrotic spots); no macroscopic symptoms are observed in the hypersensitive variety 814, which exhibits the strongest ethylene outburst 13 h after inoculation. The capacity for ethylene formation after mechanical wounding (" point freezing ") is almost identical in healthy leaves of all three varieties. This capacity is still preserved after the first ethylene outburst 36 h after infection.
and Goeschl, 1969; Abeles, 1973), released in very small amounts under physiologic conditions. During fruit ripening, flower fading, abscission, and senescence of leaves, an increased evolution of this gas is frequently observed (Abeles, 1973). An outburst of ethylene is a known phenomenon linked to mechanical and chemical injury of plants. It is also observed during the interaction between host and pathogen after infections, particularly in those complexes leading to hypersensitivity (Williamson, 1950; Ross and Williamson, 1951 ; Freebairn and Buddenhagen, 1964; Smith etal., 1964; Stahman et al., 1966; Chalutz and Devay, 1969; Balazs et al., 1969; Nakagaki et al., 1970) and seems to be associated with the necrotic process. Wheat leaves infected with Puccinia graminis tritici, or tomato plants infected with Verticillium albo atrum or with Fusarium, however, behave in the opposite way (Daly et al., 1970; Pegg and Cronshaw, 1976; Gentile and Matta, 1975) : they produce higher amounts of ethylene during the expression of susceptibility. In this communication we present data concerning ethylene evolution during the incubation cycle of Uromyces phaseoli in bean leaves susceptible and hypersensitive to the parasite in comparison to the effect of mechanical wounding (" point freezing ").
Key words: Ethylene - Hypersensitivity - Phaseolus - Rust infection - Uromyces. Materials and Methods Introduction Ethylene is known to be an endogenous regulator of growth and development in higher plants (Pratt * This work was supported by the Deutsche Forschungsgemeinschaft, by the Kleinwanzlebener Saatzucht AG (Einbeck, FRG) and by a NATO fellowship to P.M. ** Permanent address: Istituto di Patologia Vegetale, Universita Perugia, Italy *** To whom correspondance should be addressed
Bean plants (Phaseolus vulgaris L.), susceptible (var. Pinto 111) and hypersensitive (var. 814 and var. 765) to rust infection (Uromyces phaseoli), were grown in the greenhouse at 18 22~ under natural light conditions supplemented with artificial illumination (11 h photoperiod). The two primary leaves were inoculated by spraying with a suspension of uredospores, yielding saturating infection. At different times after inoculation, healthy (Fig. 1a) and infected leaf blades were carefully detached and arranged inside of plastic containers (10 cm in diameter, with ca. 20 ml volume) with rubber-sealed outlets for gas analysis. After 1, 2 and 3 h of incubation in the dark, 1 ml of the gas was analyzed as described (EIstner and Konze 1976). Ethylene concentrations were calculated
302
P. Montalbini and E.F. Elstner: Ethylene Evolution by Rust-infected Leaves
Fig. l a . Healthy leaf from Phaseolus vulgaris var. Pinto 111. b. Infected leaf from Phaseolus vulgaris var. Pinto 111, ca. 9 days after inoculation with Uromyces phaseoli. Differentiation of uredosori is visible, e. Infected leaf of Phaseolus vulgaris var. 765, ca. 9 days after inoculation with Uromyces phaseoli. Large brown lesions (but no differentiation of uredosori) are visible, d. Infected leaf of Phaseolus vulgaris var. 814, ca. 9 days after inoculation with Uromyces phaseoli. No macroscopically visible symptoms
by comparison with dilutions of authentic gas samples. Point freezing of bean leaves was performed as described (Elstner and Konze, 1976). For each 100 mg of leaf weight, 4 frozen areas were set in approximately regular distances of 1 cm, by touching a stainless steel rod with a diameter of 3 m m (kept at liquid nitrogen temperature) for 2 s on the leaf surface.
Characteristics of the Infection Within the Varieties Under the preceding conditions, flecks were very evident on Pinto 111 around 125 h after inoculation, but were barely visible 7 0 h after inoculation. Differentiation of uredosori started ca. 8-9 days after inoculation (Fig. 1 b). In variety 765, the hypersensitivity started to be visible ca. 60 h after inoculation as very small depressions of the epidermis layer, a first macroscopically visible s y m p t o m of the histologic collapse. In the subsequent 20 30 h the p h e n o m e n o n became more pronounced and ca. 100 h after the inoculation the lesions became brown coloured, indicating the end of the hypersensitivity process (Fig. 1 c). In variety 814 no symptoms of the disease are macroscopically visible. F r o m microscopic observations (Marte and Montalbini, 1972) it is known that hypersensitivity is limited to very small histologic areas. Fluorescence of the infected cells, to which collapase is closely related, appears around 2 0 h after inoculation (Fig. 1 d).
Results
I. Ethylene Production as a Response after Infection Suspensions of fungal spores as used for the infection did not produce any ethylene when kept in closed vessels for several days and there was no detectable ethylene production during spore germination on the leaves during the first 8 h after inoculation. Approximately 10 h after inoculation with a spore suspension of Uromyces phaseoli (yielding a very strong, "saturating" infection), in all three varieties (Pinto 111 = susceptible; 765 and 814=hypersensitive), an outburst of ethylene could be observed (Fig. 2). This first outburst (state 1) corresponds in timing to the production of substomatal vesicles in the stomatal cavities (cf. Mendgen and Heitefuss, 1975). In the hypersensitive variety 814 significantly more ethylene is produced than in the susceptible variety Pinto 111 or in the hypersensitive variety 765. Approximately 20 h after inoculation, this first ethylene outburst decreases and only negligible eth-
P. Montalbini and ILF. Elstner: Ethylene Evolution by Rust-infected Leaves
303
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Fig. 2. Differences in ethylene production by detached bean leaves from three varieties during the first 36 h after inoculation with Uromyces phaseoli. For experimental details see Methods. e - - e Pinto I l l (susceptible); , - - - A var. 814 (hypersensitive) ; x --- x var. 765 (hypersensitive). The points represent means (with standard error) of 12 values (4 probes from 3 independent experiments)
36
Time [hi
ylene formation is observed in all three varieties 36 h after inoculation (state 2). At 20 h after inoculation, the first haustoria of the fungal mycelium are produced in the mesophyll of the leaves (compare Fig. 3 a and 3 b).
a I0-14h after inoculation
Substornato[
~
b
ca. 20h after inoculation
Approximately 60 h after inoculation, a very strong ethylene outburst can be measured in the hypersensitive variety 765, lasting for ca. 50-60 h. This variety exhibits the lowest ethylene response during the formation of the substomatal vesicle (state 1).
c
ca, 60h after inoculation
vesicle
multiple houstoria
first haustoria
ppressorium
2s
state 1
state
2
state 3
Fig. 3. Schematic drawings of three states of fungal development on and in bean leaves (from Sempio and Caporali, 1958, and after Mendgen and Heitefuss0 I975)
304
P. Montalbini and E,F. Elstner: Ethylene Evolution by Rust-infected Leaves
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150
175
200
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Time [hi Fig. 4. Ethylene formation during the infection cycle of U r o m y c e s phaseoli in detached leaves of susceptible and hypersensitive varieties vulgaris. For experimental details see Methods. In the controls (healthy=noninfected leaves) we observed an average of P h a s e o l u s ethylene formation of below 0.5 nmol/g fresh weight x h. o - - o Pinto 111 (susceptible); A. 9 9 var. 814 (hypersensitive); x - - - x vat. 765 (hypersensitive). The points represent means (with standard error) of 12 values (4 probes from 3 independent experiments)
In the varieties Pinto 111 (susceptible) and 814 (hypersensitive) no second outburst of ethylene during this state (state 3) is observed (Fig. 4), although in the susceptible variety Pinto 111 at this time a strong mycelium is spreading throughout the mesophyll (cf. Fig. 3 c).
H. Ethylene Production as a Response after Mechanical Wounding
The ethylene/ethane ratio in the gas phase of incubated leaf disks or leaves after wounding by point freezing seems to be a good indicator for the integrity of green leaves, as far as the intactness of the compartmentalization inside the cells is concerned (Elstner and Konze, 1976). Therefore, the effect of point freezing on ethylene and ethane production by healthy and infected (states 2 and 4) bean leaves of the three varieties Pinto 111 (susceptible) and 765 and 814 (Hypersensitive) has been studied.
As shown in Table 1, all three varieties show similar responses after point freezing of parts of the leaf surface (cf. Methods), namely an approximately 7to 10-fold increase of ethylene production accompanied by ethane formation. Table 1. Effect of wounding (point freezing) on ethylene- and ethane formation by noninfected bean leaves of three varieties Variety
Untreated
After point freezing"
ethylene
ethane
ethylene
ethane
Pinto 111 (susceptible)
3.12_+3.3
0
27.52_+14.21
5.8+5.89
Var. 814 (hypersensitive)
3.12+2.8
0
32.22_+18.5
4.7_+3.08
Var. 765 (hypersensitive)
4.32+_4.9
0
31.45-+21.23
4.97-+3.52
The numbers ( x 10-11 mol/g fresh weight • h) represent means of 17 independent experiments with standard deviations (-+)
P. Montalbini and E.F. Elstner: Ethylene Evolution by Rustqnfected Leaves
305
Table 2. Effect of wounding (point freezing) on ethylene- and ethane formation by infected bean leaves of three varieties during two different states ~ of fungal development Variety
State 2
State 4
untreated
Pinto 111 (susceptible) Var. 814 (hypersensitive) Var. 765 (hypersensitive)
wounded
untreated
C2H4
C2H6
C2H4
C2H6
6.35_+0.9 5.3_+3.7 5.2_+5.3
0 0 0
41.5_+ 6.6 5.8_+ 3.6 62.5_+10.3 5.0_+ 4.7 7 9 . 0 _ + 3 3 . 8 10.3_+ 5.6
wounded
C2H4
C2H 6
C2H 4
C2H6
30.0_+22.6 20.6_+18.1 9.4_+ 9.5
0 0 0
24.3+_18.9 41.0_+16.] 75.5_+34.5
0 4.0_+4.4 9.7_+9.2
The numbers ( x 10 11 mol/g fresh weight x h) represent means of 5 independent experiments with standard deviations (_+) a The states of fungal (Uromyces phaseoli) development are explained in the text and in Figures 2 and 3
With the applied wounding technique (4 frozen leaf areas, each 3 mm in diameter/100 mg leaf weight), ethane formation on a molar basis represents ca. 10-20% of the observed ethylene formation. All three varieties also show good (although slightly different) response after wounding during state 2 (ca. 36 h after inoculation). During state 4 (ca. 125 h after inoculation) both hypersensitive varieties show a significant ethylene- and ethane response after point freezing. In the susceptible variety (Pinto 111), however, this response seems to be lost, since no increase of ethylene formation or induction of ethane formation is observed (Table 2).
Discussion
The experiments described in this communication were performed in order to study whether the plant hormone ethylene is involved in the expression of susceptibility (cf. Daly et al., 1970; Pegg and Cronshaw, 1976) or of hypersensitivity (cf. Ross and Williams, 1951; Smith et al., 1964; Balazs et al., 1969; Nakagaki et al., 1970) of bean leaves infected by Uromyces phaseoli. As shown in Figures 2 and 3, all three tested varieties (Pinto 11 l = susceptible; var. 765 and var. 814= different degrees of hypersensitivity) exhibit a pronounced ethylene outburst ca. 10 to 14 h after inoculation. This ethylene production corresponds in timing to the fungal penetration through the stomata and the formation of substomatal vesicles. In variety 814 where hypersensitivity is expressed very early (microscopic fluorescence of collapsed cells is visible ca. 18-20 h after inoculation, Marte and Montalbini, 1972), the highest ethylene outburst at this state of fungal development (state 1) is observed. The variety 765, also hypersensitive, but with very late expression of the hypersensitivity symptoms, shows only little ethylene formation at state 1, namely
ca. 25% of the response of var. 814 and ca. 50% of the response of the susceptible variety Pinto 111. There is no significantly (background of healthy leaves!) increased ethylene formation in any of the tested varieties 36 h after inoculation (state 2). In the highly resistant variety 814 and in the susceptible variety Pinto l 11 no further periods of ethylene outburst could be detected during the development of the fungal cycle. Approximately 60 h after inoculation, the variety 765 exhibits a second, but this time very strong ethylene outburst, lasting for ca. 60-65 h (state 3). After 125 h postinoculation, no further ethylene evolution significantly above background could be detected in variety 765. In order to study whether fungal penetration and development in the mesophyll of the leaves is similar to the effects observed after mechanical damage (at least as far as the ethylene response is concerned!), wounding experiments were performed during the various "nonethylene producing" states (healthy, state 2=36 h after inoculation; state 4= from 125 h after inoculation). As shown in Tables 1 and 2, all three varieties show an increased ethylene production after "point fieezing" (as a reproducible wounding technique, cf. Elstner and Konze, 1976) in healthy and infected leaves at state 2 of fungal development. There is one pronounced difference between the fungal influence and the mechanical effect on the leaves, however: after fungal infection, ethane is not detectable in any state as an accompanying gas of ethylene, while after wounding, ethane formation seems to be a characteristic feature of mechanical "decompartmentalization" of living cells (Elstner and Konze, 1976). The ability for increased ethylene production after wounding is preserved (although to various extents) in all three varieties after the ethylene outburst in state 1. In state 4, the ability for increased ethylene formation after wounding seems to be lost only in the susceptible variety Pinto 111, where very strong symptoms of infection (flecking and uredosori formation) were visible at this state.
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P. Montalbini and E.F. Elstner: Ethylene Evolution by Rust-infected Leaves
We conclude from these data that stimulation of ethylene formation after mechanical wounding and after fungal infection constitutes different processes. Since all three varieties respond to the penetration of the fungal mycelium through the stomata, but only var. 765 shows a second ethylene response due to the invasion of the mycelium in the mesophyll (state 3), we conclude that the ethylene response in state 1 and that in state 3 of vat. 765 are also different processes. The reason why there is no second ethylene outburst in var. 814 may simply be the fact that the fungus has been "killed" by the early hypersensitive reaction in state 1. The lack of ethylene response in state 3 of the susceptible variety Pinto 111 is probably not due to an exhausted (by ethylene formation in state 1) ethylene-precursor pool, since wounding of state 2 Pinto 111 leaves shows the same ethylene response as wounding of non-infected leaves. Thus either the precursors for ethylene formation during states 1 and 3 in var. 765 are different from the precursors of ethylene after wounding, or the processes of triggering the conversion of these compounds into ethylene are not the same.
References Abeles, F.B. : Ethylene in plant biology, London-New York: Academic Press 1973 Balfizs, E., G~tborjfinyi, R., Toth, A., Kirfily, Z. : Ethylene production in xanthi tobacco after systemic and local virus infections. Acta Phytopathol. Acad. Sci. Hung. 4, 355-358 (1969) Chalutz, E., De Vay, J.E. : Production of ethylene in vitro and in vivo by Ceratocystisfimbriata in relation to disease development. Phytopathology 59, 750-755 (1969)
Daly, J.M., Seevers, P.M., Ludden, P.: Studies on wheat stem rust resistance controlled at the Sr6 locusl. III. Ethylene and disease reaction. Phytopathology 60, 1648-1652 (1970) Elstner, E.F., Konze, J.R.: Effect of point freezing on ethylene and ethane production by sugar beet leaf disks. Nature 263, 351-352 (1976) Freebairn, H.T., Buddenhagen, I.W. : Ethylene production by Pseudomonas solanacearum. Nature 202, 313 314 (1964) Gentile, I.A., Matta, A. : Production of and some effects of ethylene in relation to Fusarium wilt of tomato. Physiol. Plant Pathol. 5, 27 37 (1975) Marte, M., Montalbini, P. : Microfluorescenza in foglie de fagiolo suscettibile e resistente alla ruggine. Phytopathol. Z. 75, 59-73 (1972) Mendgen, K., Heitefuss, R,: Micro-autoradiographic studies on host-parasite interactions. I: The infection of Phaseolus vulgaris with tritium labeled uredospores of Uromyces phaseoli. Arch. Microbiol. 105, 193-199 (1975) Nakagaki, Y., Hirai, T., Stahmann, M.A.: Ethylene production by detached leaves infected with tobacco mosaic virus. Virology 40, 1-9 (1970) Pegg, G.F., Cronshaw, D.K. : Ethylene production in tomato plants infected with Verticillium albo-atrum. Physiol. Plant Pathol. 8, 279-295 (1976) Pratt, H.K., Goeschl, J.D.: Physiological roles of ethylene in plants. Ann. Rev. Plant Physiol. 20, 541-584 (1969) Ross, A.F., Williamson, C.E. : Physiological active emanation from virus-infected plants. Phytopathology 41, 431438 (1951) Sempio, C., Caporali, L. : L'Uromyees Appendiculatus sul fagiolo e su altre specie: Virulenza e specializzazione. Ann. Fac. Univ. Perugia XlII, 233-277 (1958) Smith, W.H., Meigh, D.F., Parker, J.C.: Effect of damage and fungal infection on the production of ethylene by carnation. Nature 204, 92 (1964) Stahmann, M.A., Clare, B.G., Woodbury, W.: Increased disease resistance and enzyme activity induced by ethylene and ethylene production by black rot infected sweet potato tissue. Plant Physiol. 41, 1505-1512 (1966) Williamson, C.E. : Ethylene, a metabolic product of diseased or injured plants. Phytopathology 40, 205-208. (1950)
Received 3 March; accepted 1 April 1977
