Possible Role of Phytocassane, Rice Phytoalexin, in Disease Resistance of Rice Against the Blast Fungus Magnaporthe Grisea

Biosci. Biotechnol. Biochem., 67 (4), 899–902, 2003

Note Possible Role of Phytocassane, Rice Phytoalexin, in Disease Resistance of Rice against the Blast Fungus Magnaporthe grisea

Kenji UMEMURA,1,2,† Noriko OGAWA,2 Masaru SHIMURA,2 Jinichiro KOGA,1 Hideki USAMI,1 and Toshiaki KONO1

1Health & Bioscience Laboratories, Meiji Seika Kaisha Ltd., 5-3-1 Chiyoda, Sakado, Saitama 350-0289, Japan 2Plant Biological Defense System Laboratories, 1962 Sone, Nishikawa, Nishikanbara, Niigata 959-0422, Japan

Received October 3, 2002; Accepted December 27, 2002

In addition to momilactone, phytocassanes A Domannaka (both cultivars have slightly weak through E (diterpene phytoalexins) were detected in rice resistance to blast fungus disease in êlds), were leaves in êlds suŠering from rice blast. Furthermore, collected in a paddy êld in which the pathogen phytocassane accumulation was most abundant at the naturally occurred, in Akita and Yamagata Prefec- edges of necrotic lesions, indicating that the phytoalex- tures, respectively, in July 1995. Each rice leaf was ins prevent subsequent spread of the fungus from the divided into four classes according to the following infected site. In pot experiments the pattern of criteria: healthy leaves, no visible lesion or brown phytocassane accumulation in rice leaves in an incom- specks; hypersensitive response (HR)-like leaves, visi- patible interaction (infection with an avirulent race of ble brown specks; slightly withered leaves, less than Magnaporthe grisea) was more rapidly induced than in a ten necrotic lesions; dead leaves. Dead leaves of compatible interaction (infection with a virulent race of Akitakomachi were not found in the paddy êlds. M. grisea). Twenty grams of samples (about 25 leaves) were cut into pieces and then shaken with 200 ml of ethyl

Key words: phytocassane; phytoalexin; rice; disease acetate and 200 ml of 0.1 N Na2CO3 (pH 10.5) for resistance; Magnaporthe grisea 18 h. The ethyl acetate fraction was collected and then mixed with 20 ml of 0.02 N HCl and centrifuged Pathogen-induced defense mechanisms in higher at 15,000×g for 30 min. To measure the amounts plants may involve de novo synthesis of antifungal of momilactones and phytocassanes induced, the compounds, known as phytoalexins, which play an supernatant was put through high pressure liquid important role in the disease resistance of various chromatography (HPLC) using a TSKgel ODS-120T plant species.1) More than 300 structures of phytoa- column (4.6 mm i.d.×300 mm, Tosoh Corporation, lexins were isolated from approximately 900 plant Tokyo, Japan) and eluted with 45z acetonitrile. species,2) including the rice phytoalexins momilac- Phytocassanes were monitored at 280 nm and tones A and B,3) oryzalexins A through F4–7) and S,8) momilactones were monitored at 215 nm as described phytocassanes A through E 9,10) (diterpenes), and before.13) A large amount of phytocassanes and sakuranetine11) (a ‰avanone). We previously reported momilactones accumulated in HR-like and slightly that phytocassanes are induced in rice plants or cell- withered rice leaves in tested rice leaves (Table 1). suspension cultures of rice infected with pathogenic Compared with phytocassanes and momilactones, fungi or pathogen-derived elicitor and have greater su‹cient amount of oryzalexins as other rice antifungal activity against the rice blast fungus M. phytoalexins, could not be detected by our quantita- grisea, than that of other rice phytoalexins.10,12) The tive analysis. The diŠerence in total amounts of objective of this study was to examine the role of induced phytocassanes and momilactones between phytocassanes in the resistance of rice against M. HR-like and slightly withered leaves maybe a change grisea. We found that phytocassanes play an im- due to the size or the number of lesions per leaf. Fur- portant role in disease responses of rice plants. thermore, it is plausible that the content of induced Quantitative analysis of phytocassanes and phytocassanes and momilactones varied, because of momilactones in rice (Oryza sativa L. cv) leaves from diŠerences of environmental factors such as tempera- a paddy ˆeld infested with M. grisea was attempted. ture, hours of sunlight, fertility of soil, and so on. Leaves of two rice cultivars, Akitakomachi and In contrast, both phytocassanes and momilactones

† To whom correspondence should be addressed. Fax: ＋81-492-84-7598; E-mail: kenjiäumemura＠meiji.co.jp Abbreviations: M. grisea, Magnaporthe grisea; HR, hypersensitive response; HPLC, high pressure liquid chromatography; i.d., inner diameter; SA, salicylic acid; JA, jasmonic acid; PR, pathogenesis-related 900 K. UMEMURA et al.

Table 1. Amounts of Phytocassanes and Momilactones in Rice Plants from the Paddy Field

Amount of phytoalexin in rice leaves ( mgWg F.W.) Degree of Rice cultivar disease severity Phytocassane Momilactone ABCDETotalABTotal

Healthy 0.2 0.2 0.2 N.D. 0.1 0.7 2.3 0.6 2.9 Akitakomachi HR-like 3.9 1.5 0.8 0.1 0.9 7.2 5.0 3.4 8.4 Slightly withered 27.3 7.2 3.8 6.4 4.2 48.9 2.5 52.5 54.9

Healthy 0.1 0.5 0.1 0.1 0.1 0.9 3.4 1.1 4.5 HR-like 3.9 4.6 1.0 0.0 0.6 10.1 11.5 5.2 16.7 Domannaka Slightly withered 18.0 8.4 2.1 4.5 3.7 36.7 31.6 8.8 40.4 Dead 0.6 1.0 0.3 0.1 0.4 2.4 1.6 2.3 3.9

Values shown are the mean of results from triplicate assays.

Table 2. Amounts of Phytocassanes in Sections of M. grisea-infected Rice Leaves

Amount of the induced phytocassane ( mg Wg F.W.) Section ABCDE

Central zone of necrotic lesion 39.4±5.2 16±3.1 5.3±1.0 9.2±1.6 7.1±0.8 Edge of necrotic lesion 57.3±9.8 19.6±4.1 6.8±1.8 17.6±3.1 8.2±0.9 Outsideofnecroticlesion 2.4±0.3 0.8±0.1 0.3±0.1 1.1±0.2 0.2±0.1

Each value represents the mean of results from triplicate assays±SD.

each specimen (0.2 g) was measured by HPLC analysis as described. As shown in Table 2, the levels of phytocassanes were higher at the edge than in the central zone of necrotic lesions. In contrast, phytocassanes beyond the lesion were present only in small quantities. One interpretation of this result is that they might prevent the spread of subsequent fungal proliferation in the infection site. This phenomenon seems to be correlated with the HR as a plant defense mechanism. HR is characterized by the localized cell death of infected cells, accompanied by the formation of a necrotic lesion to enclose the pathogen at the infection site. HR has also been shown to be associated with coordinated inducton of salicylic acid (SA) and jasmonic acid (JA), and synthesis of pathogenesis-related (PR) proteins as well as accumulation of phytoalexins.14) Recently, various Fig. 1. The Analyzed Section of Phytocassane Accumulation in endogenous molecules, including SA and JA, have Rice Leaves. been proposed to be involved in the complex network of signaling pathways that lead to disease were hardly detected in healthy or dead leaves. These resistance.15,16) We recently reported that JA induces results may indicates nothing more than that the accumulation of phytocassanes in cell-suspension phytocassanes accumulated in infected rice leaves in cultures of rice,17) but the relationship between a paddy ˆeld, but these results also mean that these defense signal molecules and phytocassanes in HR compounds may play a role in disease resistance in mechanism remains unclear. Nevertheless, this result rice in much the same way as momilactones. suggests that phytocassanes function as an actual We then examined the site of phytocassane induc- defense response in the HR. tion in M. grisea-infected rice. The sections from Next we analyzed the pattern of phytocassane typical nectrotic lesions and non-lesion tissue of accumulation in rice leaves between an incompatible slightly withered leaves of Akitakomachi (refer to and a compatible interaction. Because some defense Table 1) were divided into three classes: the central reactions have been reported to be induced more rap- zone and edge of the lesion and beyond the lesion, as idly after inoculation in an incompatible than in a demonstrated in Fig. 1. The phytocassane content in compatible interaction,18,19) the course of phyotocas- Phytocassane in Disease Resistance of Rice 901 Several aspects of rice disease resistance cannot be explained by phytoalexins alone, but phytoalexins may contribute to disease resistance to varying degrees. DILLONS et al.21) already reported that momilactone, sakuranetin, and oryzalexin as rice phytoalexins are important factors using rice geno- types of diŠerent susceptibility to the blast fungus. Here we suggest that phytocassanes are involved in disease resistance of rice plants to the rice blast fungus M. grisea.

Acknowledgments

We thank Dr. Tomio Fukaya, Akita Agricultural Experiment Station, and Dr. Kiyohide Ishiguro, Yamagata Prefectural Agricultural Experiment Station, for providing the samples of rice plants in the paddy ˆelds and also our colleague Dr. Michiaki

Fig. 2. The Course of Phytocassane Accumulation in Rice Iwata, Meiji Seika Kaisha Ltd., for giving us valua- Leaves in Incompatible and Compatible Interactions. ble advice. After rice plants (Oryzae sativa, L cv. Akitakomachi) were challenged with a spore suspension of race 031 (race of M. grisea References avirulent to Akitakomachi) or race 007 (virulent race of M. grisea), inoculated rice leaves at given d were assayed for phytocassane with HPLC. Values shown are the mean of four 1) Kuc, J., Phytoalexins: Perspectives and prospects. In measurements. ``Mycotoxins and Phytoalexins'', eds. Sharma, R. P., and Salunkhe, D. K., CRC Press, Boca Raton, LA, pp. 595–603 (1991). sane accumulation in rice plants was examined in 2) Harborne, J. B., The comparative biochemistry of both types of interaction after inoculation. When phytoalexin induction in plants. Biochemical Sys- theˆfthleafofriceplants(Oryzae sativa,Lcv. tematics and Ecology, 27, 335–367 (1999). 3) Cartwright, D., Langcake, P., Pryce, R. J., Akitakomachi) cultivated in a phytotron was fully Leworthy,D.P.,andRide,J.P.,Chemicalactiva- expanded, the leaves were sprayed with a spore sus- tion of host defense mechanisms as a basis for crop pension of either an avirulent race 031 or a virulent protection. Nature, 267, 511–513 (1977). race 007 of M. grisea.After24hinamoistchamber 4) Akatsuka,T.,Kodama,O.,Kono,Y.,andTakeuchi, (100z humidity)at259C,thericeplantswereculti- S., 3-Hydroxy-7-oxo-sandaracopimaradiene (oryzale- vated in a phytotron alternating with 239C in light xin A), a new phytoalexin isolated from rice blast (30,000 lux) for 12 h, followed by 189Cinthedark leaves. Agric. Biol. Chem., 47, 445–447 (1983). for 12 h. Inoculated rice leaves were collected at 5) Kono, Y., Takeuchi, S., Kodama, O., and Akatsuka, given days for use in HPLC for phytocassanes as T., Absolute conˆguration of oryzalexin A and struc- described earlier. At 2 d after innoculation, brown tures of its related phytoalexins isolated from rice blast leaves infected with Pyricularia oryzae. Agric. specks were visible in both types of interaction, and Biol. Chem., 48, 253–255 (1984). then typical necrotic lesions gradually formed from 6) Sekido, H., Endo, T., Suga, R., Kodama, O., 3 d to 5 d in a compatible interaction. As shown in Akatsuka, T., Kono, Y., and Takeuchi, S., Oryzale- Fig. 2, phytocassane accumulation in rice leaves in xin D (3,7-dihydroxy-(＋)-sandaracopimaradiene), a the incompatible interaction was more rapidly in- new phytoalexin isolated from blast-infected rice duced than in the compatible interaction. This result leaves. J. Pest. Sci., 11, 369–372 (1986). suggests that the phytocassanes are somewhat related 7) Kato, T., Kodama, O., Akatsuka, T., and Hirukawa, to the disease resistance of rice against the blast fun- T., Oryzalexin E, a diterpene phytoalexin from UV- gus. The total amount of phytocassanes induced in irradiated rice leaves. Phytochemistry, 33, 79–81 both interactions was approximately the same at 7 d (1993). after fungal challenge. Therefore, rapid production 8) Kodama, O., Li, W. X., Tamogami, S., and Akatsuka, T., Oryzalexin S, a novel stemarane-type of phytocassanes may be important for disease diterpenericephytoalexin.Biosci. Biotechnol. resistance after challenge inoculation with the rice Biochem., 57, 283–287 (1992). blast fungus. However, we must take it into account 9) Koga, J., Shimura, M., Oshima, K., Ogawa, N., that in this case of an incompatible interaction the in- Yamauchi, T., and Ogasawara, N., Phytocassanes A, duction of phytocassanes might be included as local- B, C and D, novel diterpene phytoalexins from rice, ized acquired resistance (LAR) in the region sur- Oryza sativa L. Tetrahedron, 51, 7907–7918 (1995). rounding the HR.20) 10) Koga, J., Ogawa, N., Yamauchi, T., Kikuchi, M., 902 K. UMEMURA et al. Ogasawara, N., and Shimura, M., Functional moiety 17) Umemura, K., Ogawa, N., Koga, J., Iwata, M., and for the antifungal activity of phytocassane E, a diter- Usami, H., Elicitor activity of cerebroside, a sphin- pene phytoalexin from rice. Phytochemistry, 44, golipid elicitor, in cell suspension cultures of rice. 249–253 (1997). Plant Cell Physiol., 43, 778–784 (2002). 11) Kodama, O., Miyakawa, J., Akatsuka, T., and 18) Constabel, C. P., and Brisson, N., The defense-relat- Kiyosawa, S., Sakuranetin, a ‰avanone phytoalexin ed STH-2 gene product of potato shows race-speciˆc from ultraviolet-irradiated rice leaves. Phytochemis- accumulation after inoculation with low concentra- try, 31, 3807–3813 (1992). tions of Phytophthora infestans zoospores. Planta, 12) Umemura, K., Ogawa, N., Yamauchi, T., Iwata, M., 188, 289–295 (1992). Shimura, M., and Koga, J., Cerebroside elicitor 19) Hagemeiser, J., Schneider, B., Oldham, N. J., and found in diverse phytopathogens activate defense Hahlbrock, K., Accumulation of soluble and wall- responses in rice plants. Plant Cell Physiol., 41, bound indolic metabolites in Arabidopsis thaliana 676–683 (2000). leaves infected with virulent or avirulent Pseudomo- 13) Koga, J., Oshima, K., Ogawa, N., Ogasawara, N., nas syringae pathovar tomato strains. Proc. Natl. and Shimura, M., A new bioassay for measuring Acad.Sci.USA, 2, 753–758 (2001). elicitor activity in rice leaves. Ann. Phytopathol. Soc. 20) Costet, L., Cordelier, S., Dorey, S., Baillieul, F., Jpn., 64, 97–101 (1998). Fritig, B., and KauŠmann, S., Relationship between 14) Dempsy, D., Shah, J., and Klessig, D. F., Salicylic localized acquired resistance (LAR) and hypersensi- acid and disease resistance in plants. Crit. Rev. Plant tive response (HR): HR is necessary for LAR to occur Sci., 18, 547–575 (1999). and salicylic acid is not su‹cient to trigger LAR. 15) Pieterse,C.M.,andVanLoon,L.C.,Salicylicacid- Mol. Plant Microbe Interact., 12, 655–662 (1999). independent plant defense pathways. Trends Plant 21) Dillon, V. M., Overton, J., Graver, R. J., and Sci., 4, 52–58 (1999). Harborne, J. B., DiŠerences in phytoalexin response 16) Reymond, P., and Farmer, E. E., Jasmonate and among rice cultivars of diŠerent resistance to blast. salicylate as global signals for defense gene expres- Phytochemistry, 44, 599–603 (1997). sion. Curr. Opin. Plant Biol., 1, 404–411 (1998).