1368 PHYTOPATHOLOGY Mini-Review Induced Resistance for Plant Disease Control: Maximizing the Efficacy of Resistance Elicitors Dale Walters, David Walsh, Adrian Newton, and Gary Lyon First and second authors: Crop and Soil Research Group, Scottish Agricultural College, King���s Buildings, West Mains Road, Edinburgh EH9 3JG, UK and third and fourth authors: Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. Accepted for publication 15 July 2005. ABSTRACT Walters, D., Walsh, D., Newton, A., and Lyon, G. 2005. Induced resis- tance for plant disease control: Maximizing the efficacy of resistance elicitors. Phytopathology 95:1368-1373. Plants can be induced to develop enhanced resistance to pathogen infection by treatment with a variety of abiotic and biotic inducers. Biotic inducers include infection by necrotizing pathogens and plant-growth- promoting rhizobacteria, and treatment with nonpathogens or cell wall fragments. Abiotic inducers include chemicals which act at various points in the signaling pathways involved in disease resistance, as well as water stress, heat shock, and pH stress. Resistance induced by these agents (resistance elicitors) is broad spectrum and long lasting, but rarely pro- vides complete control of infection, with many resistance elicitors pro- viding between 20 and 85% disease control. There also are many reports of resistance elicitors providing no significant disease control. In the field, expression of induced resistance is likely to be influenced by the environment, genotype, and crop nutrition. Unfortunately, little informa- tion is available on the influence of these factors on expression of induced resistance. In order to maximize the efficacy of resistance elicitors, a greater understanding of these interactions is required. It also will be important to determine how induced resistance can best fit into disease control strategies because they are not, and should not be, deployed simply as ���safe fungicides���. This, in turn, will require information on the interaction of resistance elicitors with crop management practices such as appropriate-dose fungicide use. It is now well documented that treatment of plants with various agents (e.g., virulent or avirulent pathogens, nonpathogens, cell wall fragments, plant extracts, and synthetic chemicals) can lead to the induction of resistance to subsequent pathogen attack, both locally and systemically (74). This induced resistance rarely leads to complete control of pathogens following subsequent inocula- tion, but rather results in, for example, a reduction in lesion num- bers and size (34). Most notably, the expression of this induced resistance does not require the presence of major pathogen- specific resistance genes, although the defense mechanisms acti- vated are those used in other forms of plant resistance to patho- gens (21). Following application of an inducing treatment to a plant, defense mechanisms may be triggered directly or they may be triggered only once pathogen challenge has occurred (72). The defense responses activated include an oxidative burst, which can lead to cell death (21), thereby trapping the pathogen in dead cells changes in cell wall composition that can inhibit pathogen penetration and synthesis of antimicrobial compounds such as phytoalexins (20). Induction of systemic resistance can lead to the direct activation of defense-related genes, but also can lead to the priming of cells, resulting in stronger elicitation of those defenses or, indeed, other defenses following pathogen attack (11). Induced resistance can be split broadly into systemic acquired resistance (SAR) and induced systemic resistance (ISR). SAR de- velops locally or systemically in response to, for example, patho- gen infection or treatment with certain chemicals (e.g., 2,6-di- chloroisonicotinic acid [INA]) is effective against a wide range of pathogens and is mediated by a salicylic acid [SA]-dependent process (74). In contrast, ISR develops as a result of colonization of plant roots by plant-growth-promoting rhizobacteria (PGPR) and is mediated by a jasmonate- or ethylene-sensitive pathway (50). The prospect of broad-spectrum disease control using the plant���s own resistance mechanisms has led to increasing interest in the development of agents which can mimic natural inducers of resistance (74). Research has focused on the elicitor molecules released during the early stages of the plant���pathogen interaction, and on the signaling pathways used to trigger defenses locally and systemically. The elicitors examined include carbohydrate poly- mers, lipids, and glycoproteins, and are either secreted by micro- organisms or derived from the cell walls of fungi, bacteria, or plants (74) (e.g., elicitors derived from yeast cell walls) (52). Compounds which might mimic the action of SA include INA and S-methylbenzo[1,2,3]thiadiazole-7-carbothiate (acibenzolar-S- methyl) (ASM). Neither INA nor ASM possess antimicrobial activity in vitro, and they activate the same genes as does bio- logical or SA induction of resistance (33). Indeed, ASM is the first synthetic chemical developed and marketed as a SAR acti- vator and is marketed in Europe as BION and as ACTIGARD in the United States (74). INDUCED RESISTANCE AND DISEASE CONTROL: EXAMPLES FROM CONTROLLED ENVIRONMENT AND FIELD STUDIES There is a growing body of information on the efficacy of induced resistance under field conditions (70). In their review of induced resistance in conventional agriculture, Vallad and Goodman (70) highlighted 32 examples where ASM was found to provide disease control. In 28 of these studies, reductions in disease severity provided by ASM ranged from 4 to 80%, with only 3 studies reporting disease control in excess of 80% (70). ASM was marketed originally for the control of powdery mildew on wheat and barley in Europe (16) and, indeed, has been shown to reduce mildew infection on wheat by between 64 and 77% in Corresponding author: D. Walters: E-mail address: dale.walters@sac.ac.uk DOI: 10.1094 / PHYTO-95-1368 �� 2005 The American Phytopathological Society

Vol. 95, No. 12, 2005 1369 field experiments (65,66). However, some of the best levels of disease control were demonstrated on dicotyledenous crops. For example, on tobacco, ASM provided 99% control of Pseudo- monas syringae pv. tabaci, 91% control of Cercospora nicotiana, and 89% control of Alternaria alternata (10,49). They also high- lighted 60 examples where PGPR were used to control crop diseases, with reductions in disease severity of less than 80% reported in 57 of the studies (70). Particularly high levels of disease control were achieved in cucumber, with Bacillus pumilis INR-7 and Serratia marcescens 90-166 providing 86 and 89% control, respectively, of Erwinia tracheiphila (78). But what about the diverse range of other agents that have been shown to induce resistance? These include phosphates, amino acids, fatty acids, cell wall fragments, and avirulent pathogens (74) which characteristically are either far less toxic than fungi- cides or nontoxic because they do not act directly against patho- gens but entirely through the plant���s defense mechanisms. Induced resistance using phosphate salts. Dibasic and tri- basic phosphate salts were shown to induce systemic protection against anthracnose in cucumber caused by Colletotrichum lagenarium (17) and later work demonstrated the broad spectrum of disease control achieved in cucumber using phosphates (40). It was speculated that basic phosphates applied to plants could sequester apoplastic calcium, altering membrane integrity and in- fluencing the activity of apoplastic enzymes such as polygalac- turonases, thereby releasing elicitor-active oligogalacturonides from plant cell walls (17,73). Indeed, subsequent work by Orober et al. (48) showed that phosphate-mediated resistance induction in cucumber was associated with localized cell death, preceded by a rapid generation of superoxide and hydrogen peroxide. These workers also detected local and systemic increases in levels of free and conjugated SA following phosphate application (48). Recent work on barley showed that application of phosphate to first leaves reduced powdery mildew infection by 89% in second leaves (39). Application of phosphate to first leaves led to sig- nificant increases in activities of phenylalanine ammonia-lyase, peroxidase, and lipoxygenase in second leaves, and activities of these enzymes were increased further following pathogen chal- lenge (39). Phosphates also have been shown to provide disease control under field conditions. Thus, K2HPO4 applied to rice as a 50-mM spray reduced neck blast caused by the fungus Pyricu- laria oryzae by between 29 and 42%, with increases in grain yield of between 12 and 32% (36). Phosphate (K3PO4, 25 mM) applied to barley in a field trial reduced powdery mildew infection by up to 70% and gave an increase in grain yield of 12% compared with untreated controls (39). In cucumbers grown hydroponically, phosphate at 20 ppm applied to the hydroponic solution reduced powdery mildew infection by between 80 and 92%, with reduc- tions of up to 91% in numbers of conidia produced on infected leaves (53). Induced resistance using ��-aminobutyric acid. The non- protein amino acid ��-aminobutyric acid (BABA) has been shown to induce broad-spectrum resistance in a range of crops (32). BABA applied to tobacco as a 1-mM spray causes small necrotic lesions (8) and, when applied at 10 mM, led to the formation of reactive oxygen species, lipid peroxidation, induction of callose around lesions, and an increase in the SA content of leaves (61). Treatment with BABA has been reported to lead to induction of pathogenesis-related (PR) proteins. Treatment with BABA in- duced PR-1a, chitinase, and glucanase in tobacco, tomato, and pepper (9,61), but not in Arabidopsis, cauliflower, or tobacco (8,32,62). This suggests that induction of PR proteins may not be the only mode of action of BABA, which also leads to callose de- position, lignification, and hypersensitivity in some plants (9,61). BABA has been shown to move systemically in tomato, tobacco, and grape plants (9), and this may explain the systemic protection against diseases observed in these and other plants (9,61). In field trials with grapevines, BABA reduced infection by the downy mildew fungus Plasmopara viticola by 57% on cv. Chardonnay and by 98% on cv. Cabernet Sauvignon (54). Interestingly, mix- tures of BABA and fungicides were even more effective in re- ducing infection in both cultivars. Induced resistance using oligosaccharides. Oligosaccharides such as N-acetylchito-oligosaccharides and ��-1,3-glucans are well known to act as elicitors of plant defenses. Chitosan is a de- acetylated form of N-acetylchito-oligosaccharides containing poly- D-glucosamine and is a common polymer in shells of crustaceans, exoskeletons of insects, and cell walls of fungi (19). There are numerous reports of the protective effects of chitosan against pathogen infection in a range of crops. For example, chitosan seed treatment has been shown to protect tomato plants from crown rot and root rot (6) and to protect tomato seedlings against Fusarium oxysporum when applied as a foliar spray (7). A commercial formu- lation of chitosan developed by Glycogenesys Inc. (Boston), Elexa, contains 4% chitosan as its active ingredient and has been shown to protect a range of crops against important pathogens (1). In field trials with pearl millet, Elexa was shown to reduce downy mildew severity by 58% when used as a seed treatment, by 75% when used as a foliar spray, and by 77% when used as a com- bined seed treatment and foliar spray (59). Elicitors derived from the yeast Saccharomyces cerevisiae also have been shown to control plant diseases, providing up to 95% control of powdery mildew infection in barley in field trials (51,52). In that work, elicitor applied with a reduced rate of fungicide gave better disease control than the elicitor used on its own. Induced resistance using probenazole. The synthetic com- pound probenazole has been used to control rice blast caused by Magnaporthe grisea in Asia for more than 20 years and also pro- tects rice from other diseases, including bacterial blight caused by Xanthomonas oryzae pv. oryzae (31). The probenazole-containing product Oryzemate provides long-lasting control of rice blast when applied to paddy fields or to seedling boxes. It is absorbed by roots and distributed throughout the plant, and the disease control it provides lasts for up to 70 days after application (31). Importantly, despite its extensive use since the 1970s, there have been no reports of resistance development in the blast fungus (31). Early studies showed that probenazole possessed only weak antimicrobial activity, leading workers to suggest that it activated defense responses in rice (75). Indeed, recent work has confirmed this and shown that probenazole and its active metabolite 1,2- benzisothiazole-1,1-dioxide induce SAR by triggering signaling at a point upstream of SA accumulation (42). FACTORS INFLUENCING THE EXPRESSION OF INDUCED RESISTANCE From what we have considered so far, it is clear that, although there are cases where elicitors of induced resistance can provide very high levels of disease control (85%), there are many more examples of induced resistance providing lower levels of disease control (38). Indeed, there are also many reports of induced resistance not providing disease control. Thus, following a 2-year field trial with eight different cultivars of winter barley, Huth and Balke (29) concluded that ASM did not induce resistance to Barley yellow dwarf virus and, under controlled conditions, ASM did not induce resistance to Phytophthora brassicae in Arabidop- sis or P. infestans in potato (60). More recently, in an evaluation of the effects of different agents on control of X. axonopodis pv. citrumelo and X. axonopodis pv. citris on sweet oranges, ASM and the harpin protein (marketed as Messenger) failed to provide significant disease control (18). Because induced resistance is a plant response to attempted infection, it stands to reason that the expression of this response will be affected by a range of factors, including genotype and the environment. Therefore, how much do we know about the influence of these factors on the expression of induced resistance?