Biological Activities of AK-Toxins I and II, Host-Specific Toxins from Alternaria alternata Japanese Pear Pathotype*

Transcription

1 Biological Activities of AK-Toxins I and II, Host-Specific Toxins from Alternaria alternata Japanese Pear Pathotype* Hiroshi OTANI**, Keisuke KOHMOTO**, Syoyo NISHIMURA***, Tadakazu NAKASHIMA****, Tamio UENO**** and Hiroshi FUKAMI**** Abstract Pure samples of chemically characterized AK-toxins I and II from Alternaria alternata Japanese pear pathotype were examined for their biological activities. Both preparations were toxic to Japanese pear cultivars susceptible to the pathogen, but were harmless to resistant pear cultivars and non-host plants tested. AK-toxins I and II at 5 ~10-9M and 10-7M, respectively, induced characteristic veinal necrosis and rapid K+ loss on susceptible pear leaves. The dose-response relationship revealed that the activity of AK-toxin II was about one-tenth that of AK-toxin I. AK-toxins I and II at 10-4M did not affect resistant pear leaves. In susceptible pear cells treated with AK-toxin I or II, invagination of the plasma membrane and degradation of the cell wall were observed under an electron microscope. When AK-toxin I or II was added to avirulent spores of A. alternata, the spores could invade susceptible tissues, just as did the virulent spores. These results show that pure samples of AK-toxins I and II fall under the criteria of host-specific toxin, and have the same biological activity as our previous semipurified AK-toxin preparation. (Received February 1, 1985) Key Words: Alternaria alternata Japanese pear pathotype, AK-toxins I and II, plasma membrane. Introduction A distinct pathotype of Alternaria alternata (Fr.) Keissler (previously called A. kikuchiana Tanaka), causing black spot disease of Japanese pear (Pyrus serotina Rehd.), produces a host-specific toxin (AK-toxin) which selectively affects the pear cultivars that are susceptible to the fungus4,14). AK-toxin plays a key role not only in the symptom development of the disease, but also in the initial colonization in the host plants by the pathogen6,9). Therefore, AK-toxin is now recognized as a useful tool to study host-parasite interactions at the molecular level. AK-toxin causes a rapid and marked loss of electrolytes (mainly K+) from susceptible pear tissues1,8). When seen under the electron microscope, the first effect of the

2 toxin on susceptible cells is occurrence of plasma membrane modification, followed by cell wall degradation10,11). All data obtained so far support the hypothesis that the primary site of action of AK-toxin is in or on the plasma membrane of the host cells5). Toxin preparations with several different levels of purity were used in these experiments. Recently, Nakashima et al. isolated two toxins, named AK-toxins I and II, in crystalline form from culture broth of A. alternata Japanese pear pathotype2,3). The chemical structures of AK-toxins I and II were determined to be 8-(ƒ -acethylamino-ƒàmethyl-ƒà-phenyl-propionyloxy)-9, 10-epoxy-9-methyl-2E, 4Z, 6E-decatrienoic acid and its ƒà-demethyl derivative, respectively (Fig. 1). Therefore, we re-examined the evidence for the host-selective biological activities of completely purified AK-toxins I and II. A brief report of some of this work was published7). Fig. 1. Chemical structure of AK-toxins I and II. Materials and Methods Plants. Japanese pear (Pyres serotina Rehd.) cultivars susceptible (Nijisseiki) and resistant (Chojuro) to A. alternata Japanese pear pathotype were used in this study. Many other pear cultivars and non-host plants were also used in investigating the relation between susuceptibility to the pathogen and sensitivity to the toxin. Young leaves of these plants were used throughout this study. Toxin preparation. Crystalline AK-toxins I and II were prepared as described previously by Nakashima et al.2,3). These toxins were dissolved in methanol and adjusted to 10-4M with deionized water. The toxin solutions containing 5% methanol were diluted in various concentrations with deionized water. No effects of methanol were observed. Leaf-necrosis assay. Detached leaves were slightly wounded crosswise at the center of the lower surface with a needle, and the wounded sites were treated with toxin solution (50ƒÊl). The leaves were kept on sponge mats in a moist chamber at 28 C. After 24hr, the area of visible veinal necrosis was measured to estimate the degree of sensitivity to toxin. Measurement of K+ loss. Ten disks, 1.0cm in diameter, were cut from pear leaves with a leaf punch. The disks were vacuum-infiltrated with 2ml of toxin solu-

3 Ann. Phytopath. Soc. Japan 51 (3). July, tion for 20min, and were monitored for K+ loss as described previously1). The quantity of K+ leaked from the disks was measured by an atomic absorption spectrophotometer (Hitachi ). Preparation and inoculation of spores. Conidia of a highly virulent isolate (K- 103) of A. alternata Japanese pear pathotype, and an avirulent isolate (0-94) of A. alternata were produced and maintained as described previously8). Spores were washed thoroughly by centrifuging, and were suspended in deionized water. The avirulent spores were also suspended in toxin solution. Spore concentration was adjusted to 5 ~ 105 spores/ml, using Thoma's hemacytometer. The spore suspensions were sprayed on the lower surface of the detached leaves, and the leaves were placed on sponge mats in a moist chamber. After 24hr incubation at 28C, visible black necrotic spots on the leaves were scored. Light microscopy. The leaves inoculated with spores were immersed in 0.1% cotton blue in lactophenol for 2hr, and then boiled in lactophenol-ethanol solution until the chlorophyll was removed. After rinsing with fresh lactophenol-ethanol, formation of infection hyphae in the leaves was observed microscopically. Electron microscopy. Toxin-treated portions of the pear leaf tissues were trimmed with a razor blade. Procedures for preparing thin sections for electron microscopy were the same as those described previously by Park et al.12). The sections were stained with uranyl acetate and lead citrate, or alkaline bismuch12), and were observed with a transmission electron microscope (Hitachi H-500). Results Host specificity of AK-toxins I and II AK-toxins I and II at 10-5M were tested on 36 cultivars of Japanese pear differing in their susceptibility to A. alternata Japanese pear pathotype. The cultivars were highly sensitive to the pathogen and both toxins, and the other cultivars were immune (Table 1). Thus, the spectrum of toxin sensitivity was completely consistent with that of the disease susceptibility in a number of Japanese pear cultivars. AM-toxin and AF-toxin, which are host-specific toxins from apple pathotype and strawberry pathotype of A. alternata, respectively, are toxic to not only their host plants but also to some cultivars of Japanese pears5). To determine whether or not the converse relationship is true, the young leaves of apple cultivar Red Gold (sensitive to AMtoxin) and of strawberry cultivar Morioka 16 (sensitive to AF-toxin) were treated with AK-toxins I and II at 10-5M. No necrotic lesions were produced on these leaves. Furthermore, AK-toxins I and II at 10-5M were tested on about 20 species of non-host plants, such as barley, cherry, citrus, rice, rose, tobacco and tomato. In all plants tested, these toxins did not produce any visible toxicity on the leaves. Dose-response relationship of AK-toxins I and II AK-toxin I at concentrations as low as 5 ~10-9M induced veinal necrosis on leaves of susceptible cultivar Nijisseiki when droplets of the toxin solution were placed on the leaves (Fig. 2). The relationship between the toxin concentration (log scale) and leaf

4 Table 1. The correlation between susceptibility to Alternaria alternata Japanese pear pathotype and sensitivity to AK-toxins I and II among various cultivars of Japanese pear a) The reactions resulting from spore inoculation (5 ~105 spores/ml) and toxin treatment (10-5M) were evaluated as +(positive) or -(negative).

5 Ann. Phytopath. Soc. Japan 51 (3). July, Fig. 2. Relationship between the concentration of AK-toxin I or II, and formation of veinal necrosis on susceptible cv. Nijisseiki leaves. Veinal necrosis induced by AK-toxin I ( \ œ \) or II ( \ \) was measured 24hr after application of toxin solution to leaves. tissue response showed a sigmoidal curve, with the relationship being almost linear in the concentration range of 10-7 to 10-5M. In contrast, AK-toxin II was found to cause veinal necrosis on the susceptible leaves at 10-7M (Fig. 2). The dose-response study revealed that the activity of AK-toxin II was about one-tenth that of AK-toxin I. Neither toxin, even at 10-4M, caused necrosis on the resistant cultivar Chojuro. The K+ amount leaked from toxintreated susceptible and resistant leaf disks was measured. AK-toxins I and II at 10-6M caused a conspicuous increase in the loss of K+ from susceptible leaves (Fig. 3). In contrast, Fig. 3. Effects of AK-toxins I (a) and II (b) on K+ loss from pear leaf tissues. Leaf tissues of susceptible cv. Nijisseiki ( \ œ \) were treated with AK-toxin I or II at 10-6M. In resistant cv. Chojuro ( \ \), the leaves were treated with AK-toxin I or II at 10-4M. These tissues were monitored at intervals for K+ loss. Losses from susceptible ( c œ c) and resistant ( c c) controls without toxin are also shown.

6 loss of K+ from the resistant leaves was not affected by treatment with both toxins at 10-4M (Fig. 3). Plots of toxin concentration vs. either K+ loss or veinal necrosis (Fig. 4) showed essentially the same relationship. Effecf of AK-toxins I and II on the ultrastructure of cells in pear leaves Electron microscope examinations showed that 10-6M AK-toxin I caused modification of plasma membranes and degradation of cell walls in susceptible cells by 6hr after treatment (Plate Ia). Plasma membranes were invaginated and fragmented (Plate Ib). Desmotubules extended through the degraded cell walls from plasmodesmata (Plate Ic). AK-toxin II at 10-5M caused the Fig. 4. Relationship between the concentration of AK-toxin I or II, and K+ loss from susceptible cv. Nijisseiki leaves. The amount of K+ leaked from leaf tissues by AK-toxin I ( \ œ \) or II ( \ \) was estimated 4hr after toxin exposure. same changes (Plate Id). There were no effects on resistant pear leaves treated with 10-4M AK-toxin I or IT (Plate Ie). Effect of AK-toxins I and II on infection behavior of avirulent A. alternata in pear leaves Spores of avirulent A. alternata, combined with small amounts of AK-toxin I or II, were sprayed on susceptible and resistant pear leaves, and the infection behavior of spores was observed after 24hr. At concentrations of 10-8M AK-toxin I or 10-7M AKtoxin II, avirulent spores could invade susceptible tissues, and induced black necrotic spots on the leaves, just as did the virulent spores (Table 2). The rate of infection hypha formation and the number of spots increased as the concentrations of the toxins increased. However, AK-toxins I and II did not allow avirulent spores to colonize resistant tissues. Table 2. Effect of AK-toxins I and II on infection by avirulent Alternaria alternata of leaves from susceptible pear cv. Nijisseiki a) Observed at 24hr after spore inoculation (5 ~105 spores/ml).

7 Ann. Phytopath. Soc. Japan 51 (3). July, Discussion Confirmatory experiments with purified, chemically characterized AK-toxins I and II showed that the phytotoxicities were extremely active and host-selective. The sensitive range of plants to AK-toxins I and II was completely consistent with the host range to the pathogen. AK-toxins I and II had significant effects on susceptible pear tissues at 5 ~10-9M and 10-7M, respectively. Resistant pear tissues were not affected even at 10-4 M of AK-toxins I and II. These results indicate that AK-toxins I and II fall under the criteria of host-specific toxin which were proposed by Pringle and Scheffer13), and have the same biological activity as our previous semipurified AK-toxin preparation5). We have pointed out the importance of AK-toxin released from germinating spores of the pathogen6). The toxin is released from germinating spores of virulent isolates, but not released by avirulent ones9). When avirulent spores were used as inoculum together with AK-toxin, the spores could invade host tissues, indicating that the released toxin was required for initial colonization of host tissues by the pathogen9). Thus, AK-toxin is now considered to be an initiation factor for successful pathogenesis6). In the present experiments, AK-toxins I and II induced invasion of host tissues by avirulent spores. However, whether these toxins are produced by virulent spores at a key step of infection still remains to be determined. AK-toxins I and II caused rapid and drastic loss of K+ from susceptible, but not resistant, pear tissues. The first toxin-induced change in susceptible cells detected by electron microscopy was in the plasma membrane. No ultrastructural changes could be detected in the toxin-treated resistant pear cells. Our recent experiments indicated that 10-6M AK-toxin I induced a drastic decrease of membrane potential in susceptible cells within 30min after toxin exposure (unpublished work). Membrane potential in resistant cells was not affected with 5 ~10-5M AK-toxin I. Results obtained with the purified toxins confirm our previous observation that AK-toxin had a rapid effect on the plasma membrane of the host cells5). Assays for AK-toxins I and II were done by using two methods: toxin-induced leaf necrosis and K+ loss from leaves. The dose-response relationships were very similar, and a linear relationship was established between toxin concentration and host tissue response within a specified range. Thus, determinations of necrotic area and K+ loss seem to be the best assays for estimating quantity and activity of toxin. AK-toxin II is the Ĉ-demethyl derivative of AK-toxin I2,3), and has about one-tenth the activity of AK-toxin I in both assays. Further study of the structure-activity relationships of AKtoxin should be valuable in evaluating the molecular basis of toxin recognition in host cells. We wish to thank Mr. S. Yamamoto and Miss Y. Kondoh, Tottori University for their technical assistance with the electron microscope. We also wish to thank Dr. S.P. Briggs, Pioneer Hi-Bred International Inc. for critically reading the manuscript.

8 Literature cited 1. Morikawa, M., Otani, H., Nishimura, S. and Kohmoto, K. (1977). J. Fac. Agric. Tottori Univ. 12: Nakashima, T., Ueno, T. and Fukami, H. (1982). Tetrahedron Lett. 23: Nakashima, T., Ueno, T., Fukami, H., Taga, T., Masuda, H., Osaki, K., Otani, H., Kohmoto, K. and Nishimura, S. (1985). Agr. Biol. Chem. 49: Nishimura, S. (1980). Proc. Japan Acad. 56, Ser. B: Nishimura, S. and Kohmoto, K. (1983). Ann. Rev. Phytopathol. 21: Nishimura, S. and Kohmoto, K. (1983). In Toxins and Plant Pathogenesis (Daly, J.M. and Deverall, B.J. eds.). Academic Press, Sydney, pp Otani, H., Kohmoto, K., Nishimura, S., Nakashima, T., Ueno, T. and Fukami, H. (1983). Ann. Phytopath. Soc. Japan 49: 384 (Abstr.). 8. Otani, H., Nishimura, S. and Kohmoto, K. (1972). J. Fac. Agric. Tottori Univ. 7: Otani, H., Nishimura, S., Kohmoto, K., Yano, K. and Seno, T. (1975). Ann. Phytopath. Soc. Japan 41: Park, P. (1977). Physiol. Pl. Path. 11: Park, P., Fukutomi, M., Akai, S. and Nishimura, S. (1976). Ibid. 9: Park, P., Nishimura, S., Kohmoto, K., Otani, H. and Tsujimoto, K. (1981). Can. J. Bot. 59: Pringle, R.B. and Scheffer, R.P. (1964). Ann. Rev. Phytopathol. 2: Tanaka, S. (1933). Mem. Coll. Agr. Kyoto Imp. Univ. 28: Explanation of plate Plate I (a), (b), (c) Leaf cells of susceptible pear cv. Nijisseiki treated with 10-6M AK-toxin I for 6hr. (a) Invaginated plasma membrane (IP) and cell wall degradation (CWD). ~9400. (b) Membranous fragments (MF) derived from the membrane. ~ (c) Desmotubules (D) extended from plasmodesmata (PD). The section was stained with alkaline bismuth. ~ (d) Leaf cells of susceptible pear cv. Nijisseiki treated with 10-5M AK-toxin II for 6hr. AK-toxin II induced the same changes in the plasma membrane and cell wall as those of AK-toxin I. ~ (e) Leaf cells of resistant pear cv. Chojuro treated with 10-4M AK-toxin I for 24hr. No changes of ultrastructure occurred in the cells. ~5300.

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