RICE BLAST CONTROL l l PROBENAZOLE – A PLANT DEFENCE ACTIVATOR Michiaki Iwata from the Meiji Seika Kaisha Ltd. Pharmaceutical Research Centre in Yokohama in Japan discusses the mode of action of probenazole a leading agent for the control of rice blast l rice plant tissue or in an environment such as paddy water it does not reduce virulence of the fungus it does not inhibit biosynthesis of fungal melanin pigment which is essential for penetration of the fungus into the plant tissue Introduction Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-oxide) (Figure 1) is a protectant developed by Meiji Seika Kaisha Ltd. for rice blast control. Oryzemate containing probenazole has been widely used against blast by Japanese farmers since 1975 because it provides good long-lasting control when applied on a paddy field or in a seedling box.After application to rice plants probenazole is absorbed by the roots then systemically transferred to the whole plant almost completely controlling leaf blast for 40–70 days after application. Despite extensive use over many years no development of resistance in the target fungus has been observed. Figure 1. Structure of probenazole Activation of the natural plant disease defence system Most plants have the ability to escape invasion of pathogens by using defence systems even if they do not have a specific disease resistance gene. There is a delicate relationship between plant and pathogen. When environmental conditions such as temperature and humidity are favourable for the pathogen the pathogen can easily invade the plant.When the defence system of the plant functions effectively on the other hand the plant can overcome pathogen attack. Our studies show that probenazole activates the disease defence system of a plant – an unusual mode of action for a disease control chemical previously unreported. By activating the plant defence system probenazole alters the balance of the plant–pathogen relationship in favour of the plant. Non-fungicidal protection Our experiments on the effect of probenazole on the blast fungus have shown it does not have any fungicidal activity against the fungus it is not changed to a fungicidal substance within the Pest ic ide Outlook – Fe b r u a r y 2001 This journal is © The Royal Society of Chemistry 2001 l Hence although probenazole gives excellent control of blast it and its metabolites do not affect the growth or infectivity of the blast fungus.Activation of defence-related phenylpropanoid pathway Activities of enzymes in the phenylpropanoid pathway such as phenylalanine ammonia-lyase peroxidase and polyphenoloxidase are enhanced in rice plants treated with probenazole especially in plants inoculated with the blast fungus after probenazole application (Iwata et al. 1980). The phenylpropanoid pathway plays an important role in the plant defence system; when the plant is being infected lignin is synthesised and acts as a physical barrier against pathogen invasion and a phytoalexin with antimicrobial activity is produced.These contribute to the limitation of pathogen invasion in the plant tissue. Our results show that probenazole activates the phenylpropanoid pathway and thereby enhances the defence response in the plant. Accumulation of fungicidal substances We found that fungicidal substances accumulate within the tissue of the treated and inoculated rice leaf. Since probenazole and its metabolites do not have any fungicidal activity we thought that these substances originated from the rice plant. They were identified as hydroxy unsaturated fatty acids derived from a-linolenic acid (Shimura et al. 1983). We also proposed a biosynthesis pathway of these hydroxyl unsaturated fatty acids as follows a-linolenic acid cut off by phospholipase A2 from phospholipid in cell plasma membrane is peroxidized into hydroperoxylinolenic acids by lipoxygenase; then the hydroperoxides are rapidly reduced to hydroxides (Figure 2).Activities of both enzymes in the rice leaf were enhanced when the plant was inoculated with a resistant-reaction-inducing incompatible race of the blast fungus suggesting participation of both enzymes in defence response. The hydroperoxide synthesis forms part of the octadecanoid (18-carbon) pathway by which the plant hormone jasmonic acid an endogenous elicitor of defence 28 DOI 10.1039/ b100805f Figure 2. Octadecanoid cascade in plant activated by probenazole. gene expression and phytoalexin biosynthesis (Nojiri et al. 1996) is synthesized. Amplification of superoxide production Superoxide production in a protoplast prepared from rice leaves treated with probenazole was amplified by treatment with an elicitor extracted from the blast fungus cell wall showing that probenazole amplifies superoxide production in leaves attacked with the pathogen.Superoxide was released from the protoplast within several seconds after elicitor treatment suggesting that superoxide production is one of the earliest defence responses in the rice plant. In many plants production of reactive oxygen including superoxide is part of the hypersensitive response which is a powerful defence mechanism against pathogen attack (Doke et al. 1983). Since the production of reactive oxygen proceeds with rapid oxygen consumption this phenomenon is called an oxidative burst.Superoxide after generation from the NADP(H) oxidase system in plant plasma membrane is readily dismuted into hydrogen peroxide which is the most stable form of reactive oxygen. It has been reported that hydrogen peroxide is implicated in the direct killing of invading pathogen in the cross-linking of cell wall sugar proteins in the plant cell death process as a cytotoxin and in the induction of defence gene expression. Activation of the signal transduction system Plants have intercellular and intracellular signal transduction systems which transfer information from cell to cell and RICE BLAST CONTROL from outside to inside a cell relating to stresses pathogen attack wounding etc. We have observed that the defence system of the rice plant is activated through cell membrane and intracellular signal transduction pathways after treatment with a blast fungus elicitor (Kanoh et al.1993). One of the metabolites of probenazole in the rice plant accelerated an activity of cell membrane GTPase which plays an important role in membrane signal transduction from the receptor of the elicitor (Sekizawa et al. 1995). Also the expression of protein kinase C to regulate the intracellular signal transduction is induced by treatment with probenazole (Kiribuchi et al. 1998). These observations suggest that cell membrane and intracellular signal transduction systems in the rice plant are activated by probenazole. The rice plant with an activated defence signal transduction pathway can more quickly respond to the attack of pathogen and hence escape infection.Rice genes expressed by probenazole We hypothesized that the sensitization of the disease defence system in plants treated with probenazole would be brought about by a response involved with gene transcription. We screened for rice expression induced by probenazole application to prove this hypothesis and found a new rice gene PBZ1 (Midoh and Iwata 1996). The amino acid sequence estimated from the nucleic acid sequence of the PBZ1 gene showed about 30% homology with PR (pathogenesisrelated)-10 protein. This PR protein is induced after an infection of pathogen and is thought to be an infection response and defence-participating protein.When rice plants untreated with probenazole were inoculated with the blast fungus the PBZ1 gene was also induced in the rice leaf tissue. Expression of the PBZ1 gene induced by inoculation with the incompatible fungus occurred earlier than with the compatible fungus. These results show that the PBZ1 gene product is a kind of PR protein and that probenazole induces this PR protein. Expression of the PBZ1 gene was highly induced in a lesion-mimic rice mutant in which defence responses were extremely expressed (Takahashi et al. 1999). Although the function of PBZ1 protein in disease defence is still unclear the expression of the PBZ1 gene is clearly correlated with expression of disease resistance. Sakamoto et al. (1999) isolated another rice gene RPR1 (rice probenazole responsible gene) by a differential display technique.Transcription of the RPR1 gene was detected 3 days after treatment of probenazole and reached its maximum level at 6–9 days. Mode of the RPR1 expression in probenazole-treated rice plants correlated well with protection of the blast. The RPR1 protein deduced from the amino acid sequence contains a nucleotide binding site (NBS) and leucine-rich repeats (LRR). Interestingly NBS and LRR are common characteristics in the proteins coded within disease resistance genes isolated from many plants including rice. These characteristics suggest that expression of the RPR1 gene induced by probenazole leads to induction of a disease resistance response. Recently researchers have reported that many defence related genes in the rice plant are induced by application of probenazole (Shimono et al.2000; Schaffrath et al. 2000). 2 9 Pes ti cide Out look – Fe b r u a ry 2001 References RICE BLAST CONTROL Our conclusion on the mode of action of probenazole is that it prevents the invasion of pathogen by inducing many defence genes through the signal transduction pathway (Figure 3). The future Several chemicals that activate the defence system of plants like have now been reported. Benzo(1,2,3)thiadiazole-7- carbothioic acid S-methyl ester (acibenzolar-S-methyl) (Figure 4) induces systemic acquired resistance (Friedrich et al. 1996) which is one of the natural defence systems of plants. This compound has been introduced commercially by Novartis for control of a range of plant diseases.The modes of action of probenazole and acibenzolar-S-methyl are not the same because expression of the RPR1 gene is induced by acibenzolar-S-methyl but that of the PBZ1 gene is not. Figure 4. Structure of acibenzolar-S-methyl. Plant activators such as probenazole and acibenzolar-Smethyl usually do not have biocidal activity have good environmental safety are good protectants act against a wide range of plant pathogens (fungi bacteria and viruses) and have a low risk of development of pathogen resistance. It is expected that plant activators will occupy a major position as agrochemicals for controlling diseases and insects early in the 21st century. Pest ic ide Outl ook – Fe b r u a r y 2001 30 2 Figure 3.Hypothetical action site of probenazole in disease defence system of rice plant. PBZ1,RPR1 probenazoleinduced gene products; PLA phospholipase A2; LOX lipoxygenase; PAL phenylalanine ammonia-lyase; TAL tyrosine ammonia-lyase; POX peroxidase; CH2=CH2 ethylene; G GTP binding protein; PIP2 phosphatidylinositol 4,5-bisphosphate; IP3 inositol 1,4,5-triphosphate; DAG diacylglycerol; ER endoplasmic reticulum. Doke N. (1983) Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components. Physiological Plant Pathology 23 345–357. Friedrich L.; Lawton K.; Ruess W.; Masner P.; Specker N.; Rella M.G.; Meier B.; Dincher S.; Staub T.; Uknes S.; Metraux J-P.; Kessmann H.; Ryals J. (1996) A benzothiadiazole derivative induces systemic acquired resistance in tobacco. Plant Journal 10 61–70. Kanoh H.; Haga M.; Iwata M.; Sekizawa Y. (1993) Transmembrane signaling operated at rice blade cells stimulated by blast fungus elicitor I. Operation of the phospholipase C system. Journal of Pesticide Science 18 299–308. Kiribuchi K.; Dunlap D. Y.; Matsumura F.; Yamaguchi I. (1998) Protein kinase C as a biomarker for assessing the effect of environmental stress and fungal invasion on plant defence mechanism. Journal of Pesticide Science 23 123–128. Iwata M.; Suzuki Y.; Watanabe T.; Mase S.; Sekizawa Y. (1980) Effect of probenazole on the activities of enzymes related to the resistant reaction in the rice plant.Annals of the Phytopathological Society of Japan 46 297–306. Midoh N.; Iwata M. (1996) Cloning and characterization of a probenazole-inducible gene for an intracellular pathogenesisrelated protein in rice. Plant Cell Physiology 37 9–18. Nojiri H.; Sugimori M.; Yamane H.; Nishimura Y.; Yamada A.; Shibuya N.; Kodama O.; Murofushi N.; Omori T. (1996) Involvement of jasmonic acid in elicitor-induced phytoalexin production in suspension-cultured rice cells. Plant Physiology 110 387–392. Sakamoto K.; Tada Y.; Yokozeki Y.; Akagi H.; Hayashi N.; Fujimura T. Ichikawa N. (1999) Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats.Plant Molecular Biology 40 847–855. Schaffrath U.; Zabbai F.; Dudler R. (2000) Characterization of RCI-1 a chloroplastic rice lipoxygenase whose synthesis is induced by chemical plant resistance activators. European Journal of Biochemistry 267 5935-5942. Sekizawa Y.; Aoyama H.; Kimura M.; Yamaguchi I. (1995) GTPase activity in rice plasma membrane preparation enhanced by a priming effector for plant defence reactions. Journal of Pesticide Science 20 165–168. Shimono M.; Yazaki J.; Nakamura K.; Kishimoto N.; Kikuchi Takahashi A.; Kawasaki T.; Henmi K.; Shii K.; Kodama O.; Satoh H.; Shimamoto K. (1999) Lesion mimic mutants of rice with alterations in early signaling events of defence.Plant Journal 17 535–545. S.; Kubo N.; Kadowaki K.; Mochizuki A.; Yamamoto K.; Sasaki T.; Nishiguchi M. (2000) Analysis of gene expression in rice plants treated with an inducer of disease resistance probenazole using DNA microarray. . Annals of the Phytopathological Society of Japan 66 115–116 (Japanese abstract). Shimura M.; Mase S.; Iwata M.; Suzuki A.; Watanabe T.; Sekizawa Y.; Sasaki T.; Furihata K.; Seto H.; Otake N. (1983) Anti-conidial germination factors induces in the presence of probenazole in infected host leaves. III. Structural elucidation of substances A and C. Agricultural and Biological Chemistry 47 1983–1989. RICE BLAST CONTROL Michiaki Iwata is a plant pathologist at the Pharmaceutical Research Center of Meiji Seiki Kaishi Ltd.He has been involved in research into the control of plant diseases for 30 years and in particular has extensive experience in the elucidation of plant self-defence systems. 3 1 JAPAN PLANT PROTECTION ASSOCIATION The Japan Plant Protection Association (JPPA) was established in 1953 for the promotion of scientific and technical aspects of crop protection particularly with pesticides as an incorporated body under the supervision of the Ministry of Agriculture Forestry and Fisheries. Membership The membership consists of private individuals supporting members and affiliated prefectural plant protection society members (Japan is divided into 47 prefectures) Organisation The JPPA is composed of 4 divisions (General Affairs Promotion Test and Study and Publication Divisions) and a research institute.The latter operates a research and test farm at Ushiku Ibaraki Prefecture and two test farms at Noichi Kochi Prefecture and at Sadowara Miyazaki Prefecture. Activities Conferences and symposia The JPPA holds annual district plant protection conferences in 6 districts and annual symposia and field observation tours. Research and testing Here work is done firstly on research to develop and improve testing procedures to evaluate the efficacy of candidate products in controlling target pests and diseases to study effects on non-target organisms and carry out environmental fate studies. Publications Plant Protection (Shokubutsu Roueki) – monthly journal in Japanese Agrochemicals Japan – biennial journal in English and a number of books International activites The JPPA hosted the 6th International Congress of Entomology in 1980 the 5th International Congress of Pesticide Chemistry in 1982 and the 5th International Congress of Plant Pathology on 1988 (all in Kyoto). Further information Japan Plant Protection Association (JPPA) 43-11 1-chome Komagome Toshima-ku Tokyo 170 Japan. Tel. +81 (0)3 3944 1561; FAX +81 (0)3 3944 2103 Pesti cide Outlook – Fe b r u a r y 2001