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Plant–fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants
Phytochemistry 56 (2001) 253±263
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Review
Plant±fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants ReneÂe J. Grayer *, Tetsuo Kokubun Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Received in revised form 8 August 2000 Dedicated to Jerey Harborne on his retirement as editor of Phytochemistry
Abstract A brief review is given of some biological, chemical and chemotaxonomic aspects of phytoalexin research. Emphasis is placed on the search for antifungal compounds in the plant families Leguminosae and Rosaceae, and in rice, Oryza sativa. The possible role of phytoalexins in the resistance of rice plants against the fungus Pyricularia oryzae (=Magnaporthe grisea) is discussed, and the future prospects of phytoalexin research are outlined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Leguminosae; Rosaceae; Gramineae; Rice; Oryza sativa; Plant±fungal interactions; Phytoalexins; Preformed antifungal compounds
Contents 1. Introduction...........................................................................................................................................................254 2. Plant±fungal interactions .......................................................................................................................................254 3. Constitutive and induced antifungal compounds ..................................................................................................254 4. Brief history of phytoalexins..................................................................................................................................254 5. Phytoalexin research in Reading............................................................................................................................255 6. Phytoalexins and constitutive antifungal compounds in the Rosaceae..................................................................256 7. Phytoalexins in rice plants and their possible role in the resistance of rice against the rice blast pathogen, Pyricularia oryzae ..................................................................................................................................................258 8. Prospects of phytoalexin research..........................................................................................................................260 Acknowledgements.....................................................................................................................................................261 References ..................................................................................................................................................................261
* Corresponding author. Tel.:+44-20-8332-5312; fax: +44-20-8332-5310. E-mail address: [email protected] (R.J. Grayer). 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00450-7
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1. Introduction
3. Constitutive and induced antifungal compounds
In 1968 Jerey Harborne founded the Phytochemical Unit at the Department of Botany, University of Reading, with the support of the Science Research Council (UK), and the authors of this paper had the privilege to be among the numerous students, postdoctoral fellows and visitors from all ®ve continents to work in Jerey's laboratory in the last 30 years. Countless novel ¯avonoids, anthocyanins and other compounds were isolated and identi®ed in the Unit during this time, the chemotaxonomy of many plant genera and families were studied, and research was carried out on the chemical aspects of plant±insect and plant±fungal interactions. Indeed, this laboratory has been one of the leading centres for the study of induced antifungal compounds, or phytoalexins. Therefore, in recognition of Jerey's contribution to this area of study, we will give an overview of the research on phytoalexins and also constitutive antifungal compounds that took place in the laboratory at Reading from the 1970s to the present day. Some emphasis will be placed on the research projects in which we were involved ourselves, the chemical basis of resistance of rice plants against the rice blast pathogen, Pyricularia oryzae, and the antifungal compounds produced by the Rosaceae.
When a fungal spore comes in contact with a plant surface, the microclimate (temperature, humidity, light conditions, etc.) has to be right before it can germinate. Then it has to break several lines of defence set up by the plant before reaching a living cell. These include mechanical barriers such as a thick cuticle, and chemical ones such as exudate compounds which inhibit spore germination and germ tube elongation. These constituents are part of the arsenal of constitutive (or preformed) antifungal compounds produced by plants, also called preinfectional metabolites, prohibitins or phytoanticipins. If all these plant weapons are not sucient to stop germination of the fungal spore and penetration of the hyphae through the epidermis, the plant usually responds by blocking or delaying the advancement of the invader. Reactive oxygen species (ROS) are often generated as warning signals within the cell or to the neighbouring cells, triggering o various reactions (Lamb and Dixon, 1997; Wojtaszek, 1997). These include the structural reinforcement of the cell wall, the hypersensitive response (a programmed cell death), development of systemic acquired resistance (SAR) and the accumulation of newly produced antifungal chemicals which are called phytoalexins. The term phytoalexin is usually restricted to antibiotic compounds which require de novo expression of the enzymes involved in their biosynthetic pathway (Anderson, 1991). This is a very economical way to counteract pathogens, because the carbon and energy resources are diverted to phytoalexin synthesis only at the early period of infection, and only at its site. Unchallenged plants can use these resources for more basic processes of life such as development of ¯owers and production of seeds, or accumulation of reserve carbohydrates in their storage organs. Some plants do not produce phytoalexins when challenged by pathogens, but release toxins that are normally stored as less toxic glycosides in the vacuoles of their cells, e.g. phenolic and iridoid glycosides, glucosinolates and saponins (Osbourn, 1996). If the integrity of the cell is broken when penetrated by fungal hyphae, the glycoside comes into contact with hydrolysing enzymes present in other compartments of that cell, releasing the toxic aglycone. Although this aglycone released after fungal attack is not present in the intact plant and is newly produced, it is strictly speaking not a phytoalexin, because the enzymes involved (glycosidases) were already present in the healthy plant and were not formed de novo.
2. Plant±fungal interactions It is estimated that there are about 250,000 species of higher plants, but six times as many (1.5 million) species of fungi. Fungi are, ultimately, all dependent on plants for their carbon and energy source, like most other organisms that are not able to photosynthesise. Fortunately for plants, the relationship between them and fungi is usually a mutually bene®cial one. The great majority of fungi are saprophytic, i.e. they live on dead plant material, breaking this down and so recycling the nutrients to become available again for living plants. During the course of evolution, some fungi have started to interact actively with living plants. Most of these interactions are advantageous to plants, e.g. for their growth and development, as in the cases of mycorrhizae and endophytes. A small minority of fungal species has developed further and broken the ®ne balance of mutual bene®t to become plant pathogens. However, in most plant populations there are individuals that are resistant to fungal infection. The interaction between plants and their pathogens is complex and may be very speci®c to a given combination of the plant and the fungus. The defence strategies of plants against their pathogens are manifold and include the use of antifungal chemicals. On the other hand, pathogens have evolved mechanisms to evade these chemicals. The chemical aspects of the warfare between plants and fungi are discussed below.
4. Brief history of phytoalexins As long ago as 1911 the French botanist Noel Bernard discovered that plants can produce antifungal substances which are speci®cally formed when the plant
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is attacked by fungi. He found that the tubers of two orchid species, Orchis morio and Loroglossum hircinum (=Himantoglossum hircinum) became resistant to further fungal attack after they had been infected by the fungus Rhizoctonia repens (see Stoessl and Arditti, 1984). By placing infected tuber tissues on agar and introducing fungi onto the medium, Bernard found that the fungusinfected tissue produced a diusible inhibitor of fungal growth, but the compounds involved were not identi®ed until many decades later. MuÈller and BoÈrger (1940) observed the same phenomenon in potato tubers infected by Phytophthora infestans, and they called these induced substances `phytoalexins' (Greek futon=plant; alExEin=to defend). MuÈller and BoÈrger de®ned phytoalexins as ``chemical compounds produced as a result of invasion of living cells by a parasite''. This de®nition has been modi®ed frequently whenever new evidence revised earlier concepts. For instance, it soon became clear that phytoalexins were not only formed in plants after exposure to fungi, but also by various non-biological stress factors such as irradiation with short-wavelength UV light or treatment with heavy metal ions such as copper or mercury salts. For this reason Ingham (1973) rede®ned phytoalexins as ``antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors''. Others called compounds induced by environmental factors `stress compounds'. Furthermore, the term phytoalexin is generally limited to secondary metabolites of low molecular weight, usually below 1000, so that it does not apply to antifungal peptides and proteins produced by plants. It is often dicult to determine whether a compound is constitutive or induced, for the compound may normally be present in hardly detectable quantities, but dramatically increase in concentration after infection. Strictly speaking this is not a phytoalexin because it is not synthesised de novo. Therefore, Stoessl (1980) proposed a more pragmatical de®nition of phytoalexins: ``Products of higher plant metabolism, absent from healthy tissues or present only in negligible traces, which accumulate in signi®cant amounts in response to fungal or bacterial challenge''. In 1994 the new term ``phytoanticipin'' was proposed to replace and include all types of low molecular weight antifungal metabolites other than phytoalexins that are supposed to play a role in disease resistance of plants (Van Etten et al., 1994). Yet another point of confusion between constitutive and induced antifungal compounds is that the same compound may be a preformed antifungal substance in one species and a phytoalexin in another. For instance, the ¯avanone sakuranetin is a constitutive antifungal compound in the leaves of blackcurrant (Ribes nigra, Grossulariaceae) (Atkinson and Blakeman, 1982) and of Hebe cupressoides (Scrophulariaceae) (Perry and Foster, 1994), but induced in the leaves of rice (Oryza sativa, Gramineae) (Kodama et
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al., 1992a). Moreover, some compounds may be a phytoalexin in one organ and constitutive in another part of the same plant, such as momilactone A, which occurs constitutively in rice husks (Kato et al., 1973), and rice stems (Lee et al., 1999), but is a phytoalexin in rice leaves (Cartwright et al., 1981). Thus, phytoalexins are de®ned by the dynamics of biosynthesis and the functions, not by the chemical structural class they belong to, or the biosynthetic pathway through which they were formed. Phytoalexins have been found in both Gymnospermae and Angiospermae, and within the latter group in both Monocotyledoneae and Dicotyledoneae (Harborne, 1999). All the major structural classes such as phenolics, terpenoids and alkaloids, are represented, and the structures are often unique at the family level. For instance, the majority of phytoalexins produced by members of the family Leguminosae are iso¯avonoids, whereas sulfur-containing indoles are found uniquely in the family Cruciferae. However, in some other families, a variety of phytoalexins belonging to dierent compound groups are produced, e.g. in the Gramineae, Compositae and Moraceae (Grayer and Harborne, 1994; Harborne, 1999). The role that phytoalexins may play in disease resistance of plants is discussed later in this paper. 5. Phytoalexin research in Reading Jerey Harborne's ®rst PhD student, and later postdoctoral fellow, in the area of phytoalexin research, was John Ingham, who induced, isolated and identi®ed more than a hundred dierent phytoalexins from over 500 species of the Leguminosae subfamily Papilionoideae in the 1970s and 1980s. Owing to his contribution, the chemistry of phytoalexins in this group is probably better known than that of any other plant family. Ingham isolated and identi®ed no less than 53 novel iso¯avonoid structures from legumes, most of which were phytoalexins: 6 iso¯avones, 5 iso¯avanones, 1 rotenoid, 29 pterocarpans, 8 iso¯avans, 3 coumestans and 1 coumaranochromone (see Harborne and Baxter, 1999). An example is 6a-hydroxyisomedicarpin (1), a phytoalexin from the leaves of Melilotus alba (Ingham, 1976a). Some species of Leguminosae produced dierent types of phytoalexins, e.g. stilbenoids, as in the case of Arachis hypogaea hypocotyls (Ingham, 1976b). Harborne and Ingham were probably the ®rst scientists to realise the chemotaxonomic potential of phytoalexins. Good chemotaxonomic results were obtained at various levels of classi®cation, and they published a paper in Nature on the phytoalexin variation in Trigonella species (Ingham and Harborne, 1976). This laid the foundation for further chemosystematic studies on phytoalexins and preformed antifungal substances in the Leguminosae by several generations of students and also by visitors, resulting in many publications. For instance, Dave Robeson studied the phytoalexin
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response in 60 species of the genera Lathyrus, Lens, Pisum and Vicia, tribe Vicieae, of the Leguminosae. Lathyrus and Vicia are similar morphologically and had always been considered closely related, whereas Pisum and Lens were thought to be more distantly related. However, Vicia and Lens both appeared to produce furanoacetylene phytoalexins such as wyerone (2), whereas these were absent from Lathyrus and Pisum. Species of the latter genera produce pisatin (3) and related pterocarpans instead, which in turn are uncommon in Vicia and Lens. This may mean that Vicia and Lens are closely related, and Lathyrus and Pisum are close relatives, whereas Lathyrus and Vicia may be more distant than previously considered (Robeson and Harborne, 1980). More than a decade later Indrani Seneviratne did similar studies on the genus Vigna (Seneviratne and Harborne, 1992). Satoshi Tahara, a post-doctoral visitor, worked on the constitutive antifungal compounds from the genus Lupinus, because this taxon did not appear to produce phytoalexins. More than thirty novel iso¯avonoids were isolated and identi®ed (see Harborne and Baxter, 1999), e.g. lupiniso¯avone A (4) from the roots of Lupinus albus (Tahara et al., 1984). But not only the phytoalexins of the Leguminosae were studied in Reading; other students from undergraduates to PhDs have investigated the antifungal compounds in various families including the Compositae, Rosaceae and Umbelliferae. Furthermore, Jerey
Harborne's interest also extended to their potential in plant protection, either as novel fungicides or as resistance factors of crop plants against pathogenic fungi. For instance, Peter Marshall studied phytoalexins in the Compositae and also worked on plant protection. He induced phytoalexins in excised leaf or stem segments by inoculating the fungus Botrytis cinerea. The compounds involved appeared to be unstable polyacetylenes, which had to be treated with uttermost care (low temperatures and shielded from daylight). Two novel compounds were isolated from Coleostephus myconis, and these were named (E)- and (Z)-myconisols (5) (Marshall et al., 1987). The Leguminosae also yielded a potentially useful crop protection agent, a fungitoxic chromone, which was obtained as a phytoalexin from Lathyrus odoratus (Robeson et al., 1980). 6. Phytoalexins and constitutive antifungal compounds in the Rosaceae Apart from the Leguminosae, a plant family that was surveyed thoroughly in Reading for its phytoalexin response was the Rosaceae. More than 130 species were studied (Kokubun and Harborne, 1994, 1995). In all over 20 antifungal compounds were isolated and identi®ed by activity-guided fractionation, combined with a simple
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and rapid detection method (TLC-direct bioautography) (Homans and Fuchs, 1970). The family Rosaceae was chosen primarily because of its enormous economic importance, both in ornamental and in culinary terms (many fruit trees and shrubs). The reported occurrence of iso¯avonoids, although sporadic and without taxonomic value, in Prunus and Cotoneaster (see Ingham, 1983) suggested that iso¯avonoids might be distributed widely as the phytoalexins in the family. Since ten or so phytoalexins had been isolated from the family before the start of the project, it was known that phytoalexin synthesis was a feature of the family. Besides, benzoic acid (6), a phytoalexin found in the apple Bramley's Seedling, was the simplest of all phytoalexins known at the time (Brown and Swinburne, 1971), and it was expected that equally simple phytoalexins might be uncovered if the family was surveyed systematically. The Harris Garden, the botanic garden attached to the Plant Science Laboratories in Reading, contained numerous shrubby and woody species of Rosaceae, which meant that living plant material was amply available. The dynamic response of the plants, in terms of phytoalexin production, was most pronounced in the sapwood tissue of the subfamily Maloideae, where a number of variously substituted biphenyls and dibenzofurans were produced (Kokubun and Harborne, 1995). On the other hand, the wood tissue of the other three subfamilies,
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namely, Rosoideae, Spiraeoideae and Prunoideae, showed no sign of phytoalexin production at all. Instead, several constitutive antifungal metabolites were detected, but they were not isolated/identi®ed; that was out of the scope of the project. Also, their identity was somewhat predictable, i.e. ¯avanones and iso¯avones in the wood of Prunus species, tulipaline B in Spiraea (Okuma et al., 1975), and catechins and proanthocyanidins that are ubiquitous in the family, but the antifungal activity of these is only weak. Apart from the wood tissues, there were only two cases of positive phytoalexin production: in the leaves of Sorbus aucuparia, subfam. Maloideae (Kokubun and Harborne, 1994) and the roots of Sanguisorba minor, subfam. Rosoideae (Kokubun et al., 1994), where aucuparin (7) and 20 ,60 -dihydroxy-40 -methoxyacetophenone (8), respectively, were identi®ed. The latter case is the only positive result in the whole of the subfamily Rosoideae, despite a wide-ranging search for phytoalexins in naturally diseased tissue, and by arti®cial inoculation of many tissues, including leaves and fruits of strawberries and raspberries, hips and petals of roses, etc. Nevertheless, this case of positive phytoalexin synthesis may warrant a re-investigation on the alleged phytoalexin in the root of strawberry Fragaria cv. SteleMaster (Mussel and Staples, 1971), the chemical structure of which has never been worked out. The majority of the phytoalexins when they are formed appear to be biphenyls or dibenzofurans. Although they share the same C6±C6 carbon skeleton, these two types have never been observed to co-occur in the same tissue. This apparent dichotomy was most striking in the case of Eriobotrya japonica; the leaves gave the dibenzofuran eriobofuran (9) whereas the wood synthesized biphenyls such as aucuparin (7). These antifungal compounds could not be detected after acid hydrolysis of the corresponding healthy tissue, thereby con®rming their phytoalexin status. These observations could, however, well be questioned following the work carried out on cell suspension cultures of a scab-resistant apple Malusdomestica cv. Liberty (Hrazdina et al., 1997; Borejsza-Wysocki et al., 1999). These workers did not only ®nd both biphenyls and dibenzofuran in a single treatment (yeast extract), but they also isolated glycosides as well (10, 11). It would be very desirable to see whether this is also the case for dierentiated tissues, since undierentiated tissues display a somewhat unique pro®le of secondary metabolites (e.g. Monir and Proksch, 1989; Banthorpe and Brown, 1989) and may possess a glycosidation metabolism that is absent from the parent plant (e.g. Kokubo et al., 1991). In terms of chemical structures, biphenyls and dibenzofurans are rarely encountered in nature. The C6±C6 structure without alkyl substitution has been recorded so far in Umbelliferae (Williams and Harborne, 1972), Salicaceae (Malterud and Sandanger Dugstad, 1985),
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Leguminosae (Ghosal et al., 1988), Guttiferae (Cardona et al., 1990; Garcia Cortez et al., 1998), Podostemaceae (Burkhardt et al., 1992) and Verbenaceae (Cambie et al., 1997). It is interesting to note that Calophyllum pandi¯orum, a member of the Guttiferae, also produces dibenzofurans (Ito et al., 1996). As these families are taxonomically unrelated to each other, it may be that their biosynthetic pathway has evolved independently. The biosynthetic pathway leading to this structure has not been elucidated, although two dierent pathways are possible regarding the substitution pattern on the two aromatic rings: peroxidase-mediated radical coupling of simple phenols (Kobayashi et al., 1994), or the shikimateacetate/malonate pathway via stilbene synthase-like enzymes (Cotterill et al., 1974). Two recent reports on disease resistance factors of the Rosaceae may be mentioned here, although this research was not carried out at Reading. Firstly, 3,5-di-O-caeoylquinic acid was found to be the factor inhibiting the formation of the lesions in Alternaria-infected Pyrus leaves. Although this constitutive compound is not very potent as a fungicide, it is nevertheless thought to play a role in the resistance mechanism by inhibiting the formation of infection hyphae (Kodoma et al., 1998). At Reading, hydroquinone, present in pear leaves as the glycoside arbutin, was identi®ed as the principal fungicidal compound in this fruit tree (Kokubun and Harborne, 1994). This highlights further the complexity of a disease situation; a phytoalexin alone is not the sole determinant of resistance. Secondly, very recently it has been reported that unripe nectarine fruits (Prunus persica cv. Fantasia) produced seven triterpenoid antifungal compounds following mechanical damage and fungal infection (El Lahlou et al., 1999). 7. Phytoalexins in rice plants and their possible role in the resistance of rice against the rice blast pathogen, Pyricularia oryzae Rice (Oryza sativa, Gramineae) is among the world's most important staple foods. Serious diseases such as rice blast, caused by the fungus Pyricularia oryzae (syn. Magnaporthe grisea), can cause considerable losses in yield, leading to severe hardship and famine in developing countries. One way to combat these problems is to breed and grow blast-resistant rice cultivars, which is now done extensively. However, the blast resistance of a rice cultivar hardly ever lasts more than a few years, since the pathogen mutates very quickly to outwit the plant's resistance. Therefore, plant breeders continuously have to develop new resistant rice cultivars, which are selected by means of extensive and time-consuming ®eld trials. It would be much quicker and cheaper if there were simple chemical tests to ®nd out whether a new rice cultivar is resistant, e.g. determination of the phytoalexin response of
newly developed genotypes. One of the aims of our research was to see whether this is feasible, i.e. whether there is a positive correlation between phytoalexin production and resistance of a rice cultivar to the blast pathogen. The fact that phytoalexins play an important role in the disease resistance of many crop plants has now been ®rmly established (KucÂ, 1991; Hain et al., 1993; Paxton, 1994). A number of criteria have been proposed to examine whether a phytoalexin can be considered to play a role in plant defence (see Subba Rao and Strange, 1994): (1) the compound must accumulate in response to infection; (2) the compound must be inhibitory to the invading organism; (3) the compound must accumulate to inhibitory concentrations in the vicinity of the parasite at the time the parasite ceases growing; (4) varying the rate of accumulation of the phytoalexin should cause a corresponding variation in the resistance of the plant; (5) varying the sensitivity of the invading organism should cause a corresponding variation in its virulence. However, there are several aspects of plant resistance or susceptibility that cannot be explained by production of phytoalexins or other antifungal compounds alone. During the course of the coevolution between plant host and pathogens, fungi have developed their own biochemical strategies for survival. Some pathogens have evolved ways to detoxify phytoalexins by means of enzyme systems which convert these compounds to less toxic derivatives (Van Etten et al., 1989). Others have developed mechanisms to suppress the defence response of the host plant (Oku and Shiraishi, 1994), making the plant unable to perceive the pathogen entering it. This leads to failure of the hypersensitive response that is often considered to be a prerequisite for induced disease resistance including the production of phytoalexin (Dangl et al., 1996). In this case infection happens even if the plant involved is capable producing sucient amounts of phytoalexin to inhibit hyphal growth of the fungus. Leaves of rice plants produce a wide variety of both preformed and induced antifungal compounds. The preformed or constitutive antifungals include silicate (Wadham and Wynn Parry, 1990), some twelve C18 hydroxy and epoxy fatty acids based on linolenic acid, e.g. 12 and 13 (Kato et al., 1993b), and an alkylresorcinol (Susuki et al., 1996). Rice leaf phytoalexins comprise both ¯avonoid and diterpenoid constituents. Only two ¯avonoid rice phytoalexins are known, the ¯avanones sakuranetin (14, Kodama et al., 1992a) and naringenin (15, Grayer et al., 1996). Although naringenin accumulates in elicited rice leaves, strictly speaking it may not be a phytoalexin, as this compound is not only the precursor of sakuranetin, but also of most constitutive leaf ¯avonoids and therefore is likely to be a constitutive compound itself in rice leaves. Very recently a phytoalexin-speci®c O-methyltransferase was puri®ed from jasmonic acid and CuCl2-treated rice leaves which converts naringenin to the much more antifungal sakuranetin (Rakwal et al.,
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2000). The ®rst diterpenoid phytoalexins discovered in rice were momilactones A (16) and B (Cartwright et al., 1981), followed by the oryzalexins A-F and S (17) (Akatsuka et al., 1983, 1985; Kono et al., 1984; Sekido et al., 1986; Kodama et al., 1992b; Kato et al., 1993a; Tamogami et al., 1993) and four phytocassanes (e.g. phytocassane D, 18) (Koga et al., 1995). Many genes involved in rice resistance to the blast fungus have been identi®ed, and it is very likely that at least some of these genes code for a quick response by the plant's defence system and the production of high concentrations of phytoalexins. Some of the criteria proposed by Subba Rao and Strange (1994) cited above were tested to see whether phytoalexins play a role in the resistance of rice plants to the blast pathogen, using the highly resistant rice cultivar Tetep. Leaves were punch-inoculated with an avirulent strain of Pyricularia oryzae. After ®xed periods of 3±5.8 days the concentrations of the rice phytoalexins sakuranetin, oryzalexins D, E and S, and momilactone A were determined, because standard compounds were available for these (Dillon et al., 1997). Three days after inoculation, the ®rst three criteria were met by at least two of the phytoalexins produced, sakuranetin and momilactone A. To see whether higher quantities of phytoalexins are accumulated by rice genotypes with a high degree of resistance than by genotypes with a low resistance (criterion 4), a number of rice genotypes of dierent susceptibility
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to Pyricularia oryzae were tested. These were obtained from the International Rice Research Institute, The Philippines, and from the Central Rice Breeding Station, Sri Lanka. Phytoalexins were induced by means of shortwave UV irradiation of the leaves, because this method was quick and gave the most consistent results between replicates (Grayer et al., 1996). The experiments showed that there are signi®cant and consistent dierences among rice cultivars in their phytoalexin response to UV irradiation. The phytoalexins that have the highest antifungal activity (sakuranetin, momilactone A and oryzalexin S) were produced in very much higher quantities in the resistant cultivars Tetep and Ta-poo-cho-z than in the susceptible IR 50 and B 40. Indeed, B 40 did not produce any sakuranetin or oryzalexin S (Dillon et al., 1997). Of course, the phytoalexin response of rice plants to a fungus may be completely dierent from their response to UV irradiation. However, although the results of phytoalexin production by Tetep after inoculation with P. oryzae and after UV irradiation were not completely identical, they showed a similar trend. These results, and those obtained for resistant genotype Ta-poocho-z and for susceptible IR 50 and B 40 after UV irradiation, strongly suggest that phytoalexin production could be
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an important factor in the resistance of rice against the rice blast pathogen. The resistant Carreon, however, failed to produce high concentrations of phytoalexins. This cultivar showed hardly any necrosis after UV irradiation, in contrast to the other four cultivars. This may explain its low phytoalexin production. It is possible that infection with a pathogen is a more eective way of inducing phytoalexins in Carreon, but it is also possible that blast resistance in Carreon is based on dierent factors, e.g. a very impenetrable epidermis or multifunctional exudate chemicals possessing both antimicrobial activity and UV-®ltering functions. This would be worth investigating further. The results obtained with Carreon mean that some resistant rice genotypes may be overlooked in a screening programme based phytoalexin production alone, even if a pathogen is used for elicitation rather than an abiotic elicitor. Furthermore, if an abiotic elicitor such as UV-irradiation is employed, there is no guarantee that this always will result in the induction of phytoalexins, even if a biotic elicitor would have done so. Nevertheless, a biochemical approach could complement ®eld trials and oer a number of advantages. 8. Prospects of phytoalexin research Because induction of phytoalexins is now recognised as an important mechanism of plants to defend themselves against fungal pathogens, plant pathologists should be able to exploit this ability of plants to combat plant diseases. When phytoalexin research was in its infancy, there were great expectations that phytoalexins could be utilised as novel natural fungicides. So far this prospect has proved to be rather disappointing. The toxicity of many phytoalexins appears to be rather unspeci®c, so that they often are as toxic to other organisms including humans as they are to fungi. Furthermore, their fungicidal activity is generally much lower than that of commercial fungicides already on the market, and they are generally very expensive to isolate or synthesise (KucÂ, 1991). More highly active analogues may be produced, but these have the same environmental disadvantages as synthetic fungicides currently in use. As mentioned before, phytoalexins are not only induced or elicited in plants by microorganisms, but also by UV irradiation and a range of chemicals, including heavy metal salts and certain cell wall components of fungi including chitin (all agents that can induce phytoalexins are called `elicitors'). Therefore, an alternative to protect crop plants would be to spray them with environmentally harmless phytoalexin elicitors. Sinha (1994) tested a large variety of dierent elicitors to see what their eect was on the prevention of various fungal diseases in a number of crop plants and achieved good results. However, according to Kuc (1991), the repeated application of elicitors of phytoalexin accu-
mulation can cause a marked diversion of energy and carbon resources from vital processes, resulting in resistant but stunted plants. Therefore crop yields may be less than if the plants were infected! Plant resistance can also be enhanced by incorporation of genes coding for phytoalexin synthesis. For instance, the gene for stilbene synthase has been successfully transferred from the grape vine, Vitis vinifera, to tobacco plants, so that the latter produced the stilbene phytoalexin resveratrol, and acquired improved resistance to infection by Botrytis cinerea (Hain et al., 1993). However, it is relatively easy to modify plants genetically to produce resveratrol, as this only needs one enzyme (the other enzymes needed for its biosynthesis are present in most plants). It is not so easy to do the same for many other phytoalexins. For instance, for non-leguminous plants many new enzymes would be required to produce pterocarpan phytoalexins, so that many genes would have to be transferred. Furthermore, the enhancement of plant resistance by gene transfer has several undesirable aspects. If the genes coding for the production of a particular powerful phytoalexin were to be transferred to many crop plants, pathogens could develop that could break that resistance factor, e.g. by evolving enzymes to detoxify that particular phytoalexin. These could become `superbugs' and wipe out not only all the genetically modi®ed crops, but also any wild plant species that were dependent on that phytoalexin for their defence. The transfer of genes to produce a particular phytoalexin may not render a plant resistant to diseases, because it is not usually the inability of a plant to produce these compounds that makes it susceptible. The speed and magnitude of the gene expression, i.e. to produce the phytoalexins rapidly and in high enough concentrations, may be a more important factor (KucÂ, 1991). For instance, the susceptible rice genotype B 40 does not lack the ability to synthesise phytoalexins, but produces them in much lower concentrations than the resistant genotypes Tetep and Ta-poo-cho-z (Dillon et al., 1997). Therefore, a better way of utilising the knowledge of phytoalexin production in plants for crop protection is to select genotypes for their yield of induced antifungal compounds and their speed of phytoalexin response. Another way would be to stimulate the plants (even susceptible genotypes) to produce their own phytoalexins rapidly and in large quantities. This can be done by means of immunisation, i.e. by inoculation with a non-pathogenic microorganism or an incompatible strain of a pathogen. This causes a systemic resistance: after any subsequent infection with a pathogen the plant responds rapidly to accumulate phytoalexins at the site of infection and produces lignin and other products to halt the growth of the pathogen (KucÂ, 1991). For example, systemic resistance to Pyricularia oryzae in rice was induced by inoculation of the ®rst leaf with the bacterium Pseudomonas syringae pv. syringae, which
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causes a hypersensitive response in rice. Lesions caused by a subsequent inoculation with Pyricularia oryzae were dramatically decreased in number and size (Smith and MeÂtraux, 1991). This resistance may have been the result of a rapid production of phytoalexins, although this was not investigated. Apart from having various dierent potentials for agriculture, phytoalexin production is also a treasure chest for other ®elds of science, including natural product chemistry, pharmacy, ecology, biochemistry and molecular biology. For the chemist phytoalexins present ample opportunities for elucidating novel structures. Because these compounds are not usually present in healthy plants, they are not often found during standard plant screening programmes. Moreover, only some 40 out of the ca. 150 plant families of Angiospermae and Gymnospermae have been surveyed yet for their phytoalexin response (Harborne, 1999), so many new discoveries could still be made. For the pharmacist phytoalexins have the potential to show pharmacological activities, as these compounds are by de®nition antimicrobial. Interesting areas of research for biochemists are the biosynthesis and enzymes involved in the production of the myriad of phytoalexins found so far. The de novo synthesis of phytoalexins provides molecular biologists with a very convenient source for studying gene regulation mechanisms, because new expressions of genes take place in a very short time. This is a rapidly expanding area of phytoalexin research at the moment. Finally, the plant's strategy of producing phytoalexins is a worthwhile object of study for ecologists, because this has contributed to the survival of all extant plant species on which all other organisms ultimately depend. Acknowledgements We thank the publishing editor of Phytochemistry for inviting us to present this paper and members of the Plant Science Laboratories, University of Reading, for their support and continuous encouragement throughout the projects.
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Monir, H., Proksch, P., 1989. Yeast extract induced accumlation of benzofurans in cell susension cultures of Ageratina adenophora. Phytochemistry 28, 2999±3002. MuÈller, K.O., BoÈrger, H., 1940. Experimentelle Untersuchungen uÈber die Phytophthora-Resistenz der Kartoel. Arbeiten aus der Biologischen Reichsanstalt fuÈr Land- und Forstwirtschaft, Berlin-Dahlem 23, 189±231. Mussel, H.W., Staples, R.C., 1971. Phytoalexinlike compounds apparently involved in strawberry resistance to Phytophthora fragariae. Phytopathology 61, 515±517. Oku, H., Shiraishi, T., 1994. Phytoalexins and host speci®city in plant diseases. In: Daniel, M., Purkayastha, R.P. (Eds.), Handbook of Phytoalexin Metabolism and Action. Marcel Dekker, New York, pp. 41±60. Okuma, K., Ueda, A., Hashimoto, S., 1975. Methyl b,g-dihydroxya-methylene butylate, and hydroxy-a-methylene-g-butyl lactone. Chemical Abstracts 83, 40393a and 40394b. Osbourn, A.E., 1996. Preformed antimicrobial compounds and plant defense against fungal attack. The Plant Cell 8, 1821±1831. Paxton, J., 1994. Soybean phytoalexins: elicitation, nature, mode of action, and role. In: Daniel, M., Purkayastha, R.P. (Eds.), Handbook of Phytoalexin Metabolism and Action. Marcel Dekker, New York, pp. 69±83. Perry, N.B., Foster, L.M., 1994. Antiviral and antifungal ¯avonoids, plus a triterpene from Hebe cupressoides. Planta Medica 60, 491±492. Rakwal, R., Agrawal, G.K., Yonekura, M., Kodama, O., 2000. Naringenin 7-O-methyltransferase involved in the biosynthesis of the ¯avanone phytoalexin sakuranetin from rice (Oryza sativa L.). Plant Science 155, 213±221. Robeson, D.J., Harborne, J.B., 1980. A chemical dichotomy in phytoalexin induction within the tribe of Vicieae of the Leguminosae. Phytochemistry 19, 2359±2365. Robeson, D.J., Ingham, J.L., Harborne, J.B., 1980. Identi®cation of two chromone phytoalexins in the sweet pea, Lathyrus odoratus. Phytochemistry 19, 2171±2173. Sekido, H., Endo, T., Suga, R., Kodama, O., Akatsuka, T., Kono, Y., Takeuchi, S., 1986. Oryzalexin D (3,7-dihydroxy-(+)-sandaracopimaradiene), a new phytoalexin isolated from blast-infected rice leaves. Journal of Pesticide Science 11, 369±372. Seneviratne, G.I., Harborne, J.B., 1992. Constitutive ¯avonoids and induced iso¯avonoids as taxonomic markers in the genus Vigna. Biochemical Systematics and Ecology 20, 459±467. Sinha, A.K., 1994. Possible role of phytoalexin inducer chemicals in plant disease control. In: Daniel, M., Purkayastha, R.P. (Eds.), Handbook of Phytoalexin Metabolism and Action. Marcel Dekker, New York, pp. 555±591. Smith, J.A., MeÂtraux, J.-P., 1991. Pseudomonas syringae pv. syringae induces systemic resistance to Pyricularia oryzae in rice. Physiological and Molecular Plant Pathology 39, 451±461. Stoessl, A., 1980. Phytoalexins Ð a biogenetic perspective. Phytopathologische Zeitschrift 99, 251±272. Stoessl, A., Arditti, J., 1984. Orchid phytoalexins. In: Arditti, J. (Ed.), Orchid Biology. Reviews and Perspectives, III. Cornell University Press, Ithaca, pp. 151±175. Subba Rao, P.V., Strange, R.N., 1994. Chemistry, biology, and role of groundnut phytoalexins in resistance to fungal attack. In: Daniel, M., Purkayastha, R.P. (Eds.), Handbook of Phytoalexin Metabolism and Action. Marcel Dekker, New York, pp. 199±227. Susuki, Y., Esumi, Y., Hyakutake, H., Kono, Y., Sakurai, A., 1996. Isolation of 5-(80 Z-heptadecenyl)resorcinol from etiolated rice seedlings as an antifungal agent. Phytochemistry 41, 1485±1489. Tahara, S., Ingham, J.L., Nakahara, S., Mizutani, J., Harborne, J.B., 1984. Fungitoxic dihydrofuranoiso¯avones and related compounds in white lupin, Lupinus albus. Phytochemistry 23, 1889±1900. Tamogami, S., Mitani, M., Kodama, O., Akatsuka, T., 1993. Oryzalexin S, structure of a new stemarane type rice phytoalexin. Tetrahedron 49, 2025±2032.
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ReneÂe Grayer studied biology with chemistry at the University of Leiden, The Netherlands. She attained a PhD at the same university on the chemotaxonomy of the genus Veronica (Scrophulariaceae), supervised by Prof. Robert Hegnauer. In 1971 she spent 3 months in Prof. Jerey Harborne's phytochemical unit at the University of Reading to learn techniques to separate and identify ¯avonoids, and subsequently she worked in that laboratory from 1976 to 1994 on a variety of projects. These included the chemotaxonomy of the Leguminosae, ¯avonoid sweeteners from plants, and the chemical basis of resistance of crop plants to insect
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pests and fungal diseases. In 1994 she took up a position at the Royal Botanic Gardens, Kew (Biological Interactions Section, Jodrell Laboratory), where she mainly works on the chemotaxonomy of the Lamiaceae. Together with Jerey she wrote a number of research papers, review articles and chapters of books, and she also wrote many entries for the Phytochemical Dictionary and Handbook of Natural Flavonoids, both edited by Jerey Harborne and Herbert Baxter. Since 1987 she has compiled the Index to the journal Phytochemistry, taking over from Tony Swain.
Tetsuo Kokubun left his native Japan in the summer of 1991 to do a PhD with Jerey Harborne at the University of Reading, UK. The aim of his study was to ®nd phytoalexins in the Rosaceae. The Botanical Garden at the university supplied the living plant material for this study, and Jerey's enthusiasm the inspiration. This work resulted in seven papers, mostly published in this journal. In 1997, after postdoctoral positions in Auckland, New Zealand, and in Reading, he moved to the Biological Interactions Section, Jodrell Laboratory, Royal Botanic Gardens, Kew, where he now works on bioactive fungal metabolites and antimicrobial compounds from higher plants. However, he has always stayed in touch with Jerey, and recently assisted him in the compilation and editing of the Handbook of Natural Flavonoids Ð the latest compendium in the ®eld.
www.elsevier.com/locate/phytochem
Review
Plant±fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants ReneÂe J. Grayer *, Tetsuo Kokubun Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK Received in revised form 8 August 2000 Dedicated to Jerey Harborne on his retirement as editor of Phytochemistry
Abstract A brief review is given of some biological, chemical and chemotaxonomic aspects of phytoalexin research. Emphasis is placed on the search for antifungal compounds in the plant families Leguminosae and Rosaceae, and in rice, Oryza sativa. The possible role of phytoalexins in the resistance of rice plants against the fungus Pyricularia oryzae (=Magnaporthe grisea) is discussed, and the future prospects of phytoalexin research are outlined. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Leguminosae; Rosaceae; Gramineae; Rice; Oryza sativa; Plant±fungal interactions; Phytoalexins; Preformed antifungal compounds
Contents 1. Introduction...........................................................................................................................................................254 2. Plant±fungal interactions .......................................................................................................................................254 3. Constitutive and induced antifungal compounds ..................................................................................................254 4. Brief history of phytoalexins..................................................................................................................................254 5. Phytoalexin research in Reading............................................................................................................................255 6. Phytoalexins and constitutive antifungal compounds in the Rosaceae..................................................................256 7. Phytoalexins in rice plants and their possible role in the resistance of rice against the rice blast pathogen, Pyricularia oryzae ..................................................................................................................................................258 8. Prospects of phytoalexin research..........................................................................................................................260 Acknowledgements.....................................................................................................................................................261 References ..................................................................................................................................................................261
* Corresponding author. Tel.:+44-20-8332-5312; fax: +44-20-8332-5310. E-mail address: [email protected] (R.J. Grayer). 0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00450-7
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1. Introduction
3. Constitutive and induced antifungal compounds
In 1968 Jerey Harborne founded the Phytochemical Unit at the Department of Botany, University of Reading, with the support of the Science Research Council (UK), and the authors of this paper had the privilege to be among the numerous students, postdoctoral fellows and visitors from all ®ve continents to work in Jerey's laboratory in the last 30 years. Countless novel ¯avonoids, anthocyanins and other compounds were isolated and identi®ed in the Unit during this time, the chemotaxonomy of many plant genera and families were studied, and research was carried out on the chemical aspects of plant±insect and plant±fungal interactions. Indeed, this laboratory has been one of the leading centres for the study of induced antifungal compounds, or phytoalexins. Therefore, in recognition of Jerey's contribution to this area of study, we will give an overview of the research on phytoalexins and also constitutive antifungal compounds that took place in the laboratory at Reading from the 1970s to the present day. Some emphasis will be placed on the research projects in which we were involved ourselves, the chemical basis of resistance of rice plants against the rice blast pathogen, Pyricularia oryzae, and the antifungal compounds produced by the Rosaceae.
When a fungal spore comes in contact with a plant surface, the microclimate (temperature, humidity, light conditions, etc.) has to be right before it can germinate. Then it has to break several lines of defence set up by the plant before reaching a living cell. These include mechanical barriers such as a thick cuticle, and chemical ones such as exudate compounds which inhibit spore germination and germ tube elongation. These constituents are part of the arsenal of constitutive (or preformed) antifungal compounds produced by plants, also called preinfectional metabolites, prohibitins or phytoanticipins. If all these plant weapons are not sucient to stop germination of the fungal spore and penetration of the hyphae through the epidermis, the plant usually responds by blocking or delaying the advancement of the invader. Reactive oxygen species (ROS) are often generated as warning signals within the cell or to the neighbouring cells, triggering o various reactions (Lamb and Dixon, 1997; Wojtaszek, 1997). These include the structural reinforcement of the cell wall, the hypersensitive response (a programmed cell death), development of systemic acquired resistance (SAR) and the accumulation of newly produced antifungal chemicals which are called phytoalexins. The term phytoalexin is usually restricted to antibiotic compounds which require de novo expression of the enzymes involved in their biosynthetic pathway (Anderson, 1991). This is a very economical way to counteract pathogens, because the carbon and energy resources are diverted to phytoalexin synthesis only at the early period of infection, and only at its site. Unchallenged plants can use these resources for more basic processes of life such as development of ¯owers and production of seeds, or accumulation of reserve carbohydrates in their storage organs. Some plants do not produce phytoalexins when challenged by pathogens, but release toxins that are normally stored as less toxic glycosides in the vacuoles of their cells, e.g. phenolic and iridoid glycosides, glucosinolates and saponins (Osbourn, 1996). If the integrity of the cell is broken when penetrated by fungal hyphae, the glycoside comes into contact with hydrolysing enzymes present in other compartments of that cell, releasing the toxic aglycone. Although this aglycone released after fungal attack is not present in the intact plant and is newly produced, it is strictly speaking not a phytoalexin, because the enzymes involved (glycosidases) were already present in the healthy plant and were not formed de novo.
2. Plant±fungal interactions It is estimated that there are about 250,000 species of higher plants, but six times as many (1.5 million) species of fungi. Fungi are, ultimately, all dependent on plants for their carbon and energy source, like most other organisms that are not able to photosynthesise. Fortunately for plants, the relationship between them and fungi is usually a mutually bene®cial one. The great majority of fungi are saprophytic, i.e. they live on dead plant material, breaking this down and so recycling the nutrients to become available again for living plants. During the course of evolution, some fungi have started to interact actively with living plants. Most of these interactions are advantageous to plants, e.g. for their growth and development, as in the cases of mycorrhizae and endophytes. A small minority of fungal species has developed further and broken the ®ne balance of mutual bene®t to become plant pathogens. However, in most plant populations there are individuals that are resistant to fungal infection. The interaction between plants and their pathogens is complex and may be very speci®c to a given combination of the plant and the fungus. The defence strategies of plants against their pathogens are manifold and include the use of antifungal chemicals. On the other hand, pathogens have evolved mechanisms to evade these chemicals. The chemical aspects of the warfare between plants and fungi are discussed below.
4. Brief history of phytoalexins As long ago as 1911 the French botanist Noel Bernard discovered that plants can produce antifungal substances which are speci®cally formed when the plant
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is attacked by fungi. He found that the tubers of two orchid species, Orchis morio and Loroglossum hircinum (=Himantoglossum hircinum) became resistant to further fungal attack after they had been infected by the fungus Rhizoctonia repens (see Stoessl and Arditti, 1984). By placing infected tuber tissues on agar and introducing fungi onto the medium, Bernard found that the fungusinfected tissue produced a diusible inhibitor of fungal growth, but the compounds involved were not identi®ed until many decades later. MuÈller and BoÈrger (1940) observed the same phenomenon in potato tubers infected by Phytophthora infestans, and they called these induced substances `phytoalexins' (Greek futon=plant; alExEin=to defend). MuÈller and BoÈrger de®ned phytoalexins as ``chemical compounds produced as a result of invasion of living cells by a parasite''. This de®nition has been modi®ed frequently whenever new evidence revised earlier concepts. For instance, it soon became clear that phytoalexins were not only formed in plants after exposure to fungi, but also by various non-biological stress factors such as irradiation with short-wavelength UV light or treatment with heavy metal ions such as copper or mercury salts. For this reason Ingham (1973) rede®ned phytoalexins as ``antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors''. Others called compounds induced by environmental factors `stress compounds'. Furthermore, the term phytoalexin is generally limited to secondary metabolites of low molecular weight, usually below 1000, so that it does not apply to antifungal peptides and proteins produced by plants. It is often dicult to determine whether a compound is constitutive or induced, for the compound may normally be present in hardly detectable quantities, but dramatically increase in concentration after infection. Strictly speaking this is not a phytoalexin because it is not synthesised de novo. Therefore, Stoessl (1980) proposed a more pragmatical de®nition of phytoalexins: ``Products of higher plant metabolism, absent from healthy tissues or present only in negligible traces, which accumulate in signi®cant amounts in response to fungal or bacterial challenge''. In 1994 the new term ``phytoanticipin'' was proposed to replace and include all types of low molecular weight antifungal metabolites other than phytoalexins that are supposed to play a role in disease resistance of plants (Van Etten et al., 1994). Yet another point of confusion between constitutive and induced antifungal compounds is that the same compound may be a preformed antifungal substance in one species and a phytoalexin in another. For instance, the ¯avanone sakuranetin is a constitutive antifungal compound in the leaves of blackcurrant (Ribes nigra, Grossulariaceae) (Atkinson and Blakeman, 1982) and of Hebe cupressoides (Scrophulariaceae) (Perry and Foster, 1994), but induced in the leaves of rice (Oryza sativa, Gramineae) (Kodama et
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al., 1992a). Moreover, some compounds may be a phytoalexin in one organ and constitutive in another part of the same plant, such as momilactone A, which occurs constitutively in rice husks (Kato et al., 1973), and rice stems (Lee et al., 1999), but is a phytoalexin in rice leaves (Cartwright et al., 1981). Thus, phytoalexins are de®ned by the dynamics of biosynthesis and the functions, not by the chemical structural class they belong to, or the biosynthetic pathway through which they were formed. Phytoalexins have been found in both Gymnospermae and Angiospermae, and within the latter group in both Monocotyledoneae and Dicotyledoneae (Harborne, 1999). All the major structural classes such as phenolics, terpenoids and alkaloids, are represented, and the structures are often unique at the family level. For instance, the majority of phytoalexins produced by members of the family Leguminosae are iso¯avonoids, whereas sulfur-containing indoles are found uniquely in the family Cruciferae. However, in some other families, a variety of phytoalexins belonging to dierent compound groups are produced, e.g. in the Gramineae, Compositae and Moraceae (Grayer and Harborne, 1994; Harborne, 1999). The role that phytoalexins may play in disease resistance of plants is discussed later in this paper. 5. Phytoalexin research in Reading Jerey Harborne's ®rst PhD student, and later postdoctoral fellow, in the area of phytoalexin research, was John Ingham, who induced, isolated and identi®ed more than a hundred dierent phytoalexins from over 500 species of the Leguminosae subfamily Papilionoideae in the 1970s and 1980s. Owing to his contribution, the chemistry of phytoalexins in this group is probably better known than that of any other plant family. Ingham isolated and identi®ed no less than 53 novel iso¯avonoid structures from legumes, most of which were phytoalexins: 6 iso¯avones, 5 iso¯avanones, 1 rotenoid, 29 pterocarpans, 8 iso¯avans, 3 coumestans and 1 coumaranochromone (see Harborne and Baxter, 1999). An example is 6a-hydroxyisomedicarpin (1), a phytoalexin from the leaves of Melilotus alba (Ingham, 1976a). Some species of Leguminosae produced dierent types of phytoalexins, e.g. stilbenoids, as in the case of Arachis hypogaea hypocotyls (Ingham, 1976b). Harborne and Ingham were probably the ®rst scientists to realise the chemotaxonomic potential of phytoalexins. Good chemotaxonomic results were obtained at various levels of classi®cation, and they published a paper in Nature on the phytoalexin variation in Trigonella species (Ingham and Harborne, 1976). This laid the foundation for further chemosystematic studies on phytoalexins and preformed antifungal substances in the Leguminosae by several generations of students and also by visitors, resulting in many publications. For instance, Dave Robeson studied the phytoalexin
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response in 60 species of the genera Lathyrus, Lens, Pisum and Vicia, tribe Vicieae, of the Leguminosae. Lathyrus and Vicia are similar morphologically and had always been considered closely related, whereas Pisum and Lens were thought to be more distantly related. However, Vicia and Lens both appeared to produce furanoacetylene phytoalexins such as wyerone (2), whereas these were absent from Lathyrus and Pisum. Species of the latter genera produce pisatin (3) and related pterocarpans instead, which in turn are uncommon in Vicia and Lens. This may mean that Vicia and Lens are closely related, and Lathyrus and Pisum are close relatives, whereas Lathyrus and Vicia may be more distant than previously considered (Robeson and Harborne, 1980). More than a decade later Indrani Seneviratne did similar studies on the genus Vigna (Seneviratne and Harborne, 1992). Satoshi Tahara, a post-doctoral visitor, worked on the constitutive antifungal compounds from the genus Lupinus, because this taxon did not appear to produce phytoalexins. More than thirty novel iso¯avonoids were isolated and identi®ed (see Harborne and Baxter, 1999), e.g. lupiniso¯avone A (4) from the roots of Lupinus albus (Tahara et al., 1984). But not only the phytoalexins of the Leguminosae were studied in Reading; other students from undergraduates to PhDs have investigated the antifungal compounds in various families including the Compositae, Rosaceae and Umbelliferae. Furthermore, Jerey
Harborne's interest also extended to their potential in plant protection, either as novel fungicides or as resistance factors of crop plants against pathogenic fungi. For instance, Peter Marshall studied phytoalexins in the Compositae and also worked on plant protection. He induced phytoalexins in excised leaf or stem segments by inoculating the fungus Botrytis cinerea. The compounds involved appeared to be unstable polyacetylenes, which had to be treated with uttermost care (low temperatures and shielded from daylight). Two novel compounds were isolated from Coleostephus myconis, and these were named (E)- and (Z)-myconisols (5) (Marshall et al., 1987). The Leguminosae also yielded a potentially useful crop protection agent, a fungitoxic chromone, which was obtained as a phytoalexin from Lathyrus odoratus (Robeson et al., 1980). 6. Phytoalexins and constitutive antifungal compounds in the Rosaceae Apart from the Leguminosae, a plant family that was surveyed thoroughly in Reading for its phytoalexin response was the Rosaceae. More than 130 species were studied (Kokubun and Harborne, 1994, 1995). In all over 20 antifungal compounds were isolated and identi®ed by activity-guided fractionation, combined with a simple
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and rapid detection method (TLC-direct bioautography) (Homans and Fuchs, 1970). The family Rosaceae was chosen primarily because of its enormous economic importance, both in ornamental and in culinary terms (many fruit trees and shrubs). The reported occurrence of iso¯avonoids, although sporadic and without taxonomic value, in Prunus and Cotoneaster (see Ingham, 1983) suggested that iso¯avonoids might be distributed widely as the phytoalexins in the family. Since ten or so phytoalexins had been isolated from the family before the start of the project, it was known that phytoalexin synthesis was a feature of the family. Besides, benzoic acid (6), a phytoalexin found in the apple Bramley's Seedling, was the simplest of all phytoalexins known at the time (Brown and Swinburne, 1971), and it was expected that equally simple phytoalexins might be uncovered if the family was surveyed systematically. The Harris Garden, the botanic garden attached to the Plant Science Laboratories in Reading, contained numerous shrubby and woody species of Rosaceae, which meant that living plant material was amply available. The dynamic response of the plants, in terms of phytoalexin production, was most pronounced in the sapwood tissue of the subfamily Maloideae, where a number of variously substituted biphenyls and dibenzofurans were produced (Kokubun and Harborne, 1995). On the other hand, the wood tissue of the other three subfamilies,
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namely, Rosoideae, Spiraeoideae and Prunoideae, showed no sign of phytoalexin production at all. Instead, several constitutive antifungal metabolites were detected, but they were not isolated/identi®ed; that was out of the scope of the project. Also, their identity was somewhat predictable, i.e. ¯avanones and iso¯avones in the wood of Prunus species, tulipaline B in Spiraea (Okuma et al., 1975), and catechins and proanthocyanidins that are ubiquitous in the family, but the antifungal activity of these is only weak. Apart from the wood tissues, there were only two cases of positive phytoalexin production: in the leaves of Sorbus aucuparia, subfam. Maloideae (Kokubun and Harborne, 1994) and the roots of Sanguisorba minor, subfam. Rosoideae (Kokubun et al., 1994), where aucuparin (7) and 20 ,60 -dihydroxy-40 -methoxyacetophenone (8), respectively, were identi®ed. The latter case is the only positive result in the whole of the subfamily Rosoideae, despite a wide-ranging search for phytoalexins in naturally diseased tissue, and by arti®cial inoculation of many tissues, including leaves and fruits of strawberries and raspberries, hips and petals of roses, etc. Nevertheless, this case of positive phytoalexin synthesis may warrant a re-investigation on the alleged phytoalexin in the root of strawberry Fragaria cv. SteleMaster (Mussel and Staples, 1971), the chemical structure of which has never been worked out. The majority of the phytoalexins when they are formed appear to be biphenyls or dibenzofurans. Although they share the same C6±C6 carbon skeleton, these two types have never been observed to co-occur in the same tissue. This apparent dichotomy was most striking in the case of Eriobotrya japonica; the leaves gave the dibenzofuran eriobofuran (9) whereas the wood synthesized biphenyls such as aucuparin (7). These antifungal compounds could not be detected after acid hydrolysis of the corresponding healthy tissue, thereby con®rming their phytoalexin status. These observations could, however, well be questioned following the work carried out on cell suspension cultures of a scab-resistant apple Malusdomestica cv. Liberty (Hrazdina et al., 1997; Borejsza-Wysocki et al., 1999). These workers did not only ®nd both biphenyls and dibenzofuran in a single treatment (yeast extract), but they also isolated glycosides as well (10, 11). It would be very desirable to see whether this is also the case for dierentiated tissues, since undierentiated tissues display a somewhat unique pro®le of secondary metabolites (e.g. Monir and Proksch, 1989; Banthorpe and Brown, 1989) and may possess a glycosidation metabolism that is absent from the parent plant (e.g. Kokubo et al., 1991). In terms of chemical structures, biphenyls and dibenzofurans are rarely encountered in nature. The C6±C6 structure without alkyl substitution has been recorded so far in Umbelliferae (Williams and Harborne, 1972), Salicaceae (Malterud and Sandanger Dugstad, 1985),
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Leguminosae (Ghosal et al., 1988), Guttiferae (Cardona et al., 1990; Garcia Cortez et al., 1998), Podostemaceae (Burkhardt et al., 1992) and Verbenaceae (Cambie et al., 1997). It is interesting to note that Calophyllum pandi¯orum, a member of the Guttiferae, also produces dibenzofurans (Ito et al., 1996). As these families are taxonomically unrelated to each other, it may be that their biosynthetic pathway has evolved independently. The biosynthetic pathway leading to this structure has not been elucidated, although two dierent pathways are possible regarding the substitution pattern on the two aromatic rings: peroxidase-mediated radical coupling of simple phenols (Kobayashi et al., 1994), or the shikimateacetate/malonate pathway via stilbene synthase-like enzymes (Cotterill et al., 1974). Two recent reports on disease resistance factors of the Rosaceae may be mentioned here, although this research was not carried out at Reading. Firstly, 3,5-di-O-caeoylquinic acid was found to be the factor inhibiting the formation of the lesions in Alternaria-infected Pyrus leaves. Although this constitutive compound is not very potent as a fungicide, it is nevertheless thought to play a role in the resistance mechanism by inhibiting the formation of infection hyphae (Kodoma et al., 1998). At Reading, hydroquinone, present in pear leaves as the glycoside arbutin, was identi®ed as the principal fungicidal compound in this fruit tree (Kokubun and Harborne, 1994). This highlights further the complexity of a disease situation; a phytoalexin alone is not the sole determinant of resistance. Secondly, very recently it has been reported that unripe nectarine fruits (Prunus persica cv. Fantasia) produced seven triterpenoid antifungal compounds following mechanical damage and fungal infection (El Lahlou et al., 1999). 7. Phytoalexins in rice plants and their possible role in the resistance of rice against the rice blast pathogen, Pyricularia oryzae Rice (Oryza sativa, Gramineae) is among the world's most important staple foods. Serious diseases such as rice blast, caused by the fungus Pyricularia oryzae (syn. Magnaporthe grisea), can cause considerable losses in yield, leading to severe hardship and famine in developing countries. One way to combat these problems is to breed and grow blast-resistant rice cultivars, which is now done extensively. However, the blast resistance of a rice cultivar hardly ever lasts more than a few years, since the pathogen mutates very quickly to outwit the plant's resistance. Therefore, plant breeders continuously have to develop new resistant rice cultivars, which are selected by means of extensive and time-consuming ®eld trials. It would be much quicker and cheaper if there were simple chemical tests to ®nd out whether a new rice cultivar is resistant, e.g. determination of the phytoalexin response of
newly developed genotypes. One of the aims of our research was to see whether this is feasible, i.e. whether there is a positive correlation between phytoalexin production and resistance of a rice cultivar to the blast pathogen. The fact that phytoalexins play an important role in the disease resistance of many crop plants has now been ®rmly established (KucÂ, 1991; Hain et al., 1993; Paxton, 1994). A number of criteria have been proposed to examine whether a phytoalexin can be considered to play a role in plant defence (see Subba Rao and Strange, 1994): (1) the compound must accumulate in response to infection; (2) the compound must be inhibitory to the invading organism; (3) the compound must accumulate to inhibitory concentrations in the vicinity of the parasite at the time the parasite ceases growing; (4) varying the rate of accumulation of the phytoalexin should cause a corresponding variation in the resistance of the plant; (5) varying the sensitivity of the invading organism should cause a corresponding variation in its virulence. However, there are several aspects of plant resistance or susceptibility that cannot be explained by production of phytoalexins or other antifungal compounds alone. During the course of the coevolution between plant host and pathogens, fungi have developed their own biochemical strategies for survival. Some pathogens have evolved ways to detoxify phytoalexins by means of enzyme systems which convert these compounds to less toxic derivatives (Van Etten et al., 1989). Others have developed mechanisms to suppress the defence response of the host plant (Oku and Shiraishi, 1994), making the plant unable to perceive the pathogen entering it. This leads to failure of the hypersensitive response that is often considered to be a prerequisite for induced disease resistance including the production of phytoalexin (Dangl et al., 1996). In this case infection happens even if the plant involved is capable producing sucient amounts of phytoalexin to inhibit hyphal growth of the fungus. Leaves of rice plants produce a wide variety of both preformed and induced antifungal compounds. The preformed or constitutive antifungals include silicate (Wadham and Wynn Parry, 1990), some twelve C18 hydroxy and epoxy fatty acids based on linolenic acid, e.g. 12 and 13 (Kato et al., 1993b), and an alkylresorcinol (Susuki et al., 1996). Rice leaf phytoalexins comprise both ¯avonoid and diterpenoid constituents. Only two ¯avonoid rice phytoalexins are known, the ¯avanones sakuranetin (14, Kodama et al., 1992a) and naringenin (15, Grayer et al., 1996). Although naringenin accumulates in elicited rice leaves, strictly speaking it may not be a phytoalexin, as this compound is not only the precursor of sakuranetin, but also of most constitutive leaf ¯avonoids and therefore is likely to be a constitutive compound itself in rice leaves. Very recently a phytoalexin-speci®c O-methyltransferase was puri®ed from jasmonic acid and CuCl2-treated rice leaves which converts naringenin to the much more antifungal sakuranetin (Rakwal et al.,
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2000). The ®rst diterpenoid phytoalexins discovered in rice were momilactones A (16) and B (Cartwright et al., 1981), followed by the oryzalexins A-F and S (17) (Akatsuka et al., 1983, 1985; Kono et al., 1984; Sekido et al., 1986; Kodama et al., 1992b; Kato et al., 1993a; Tamogami et al., 1993) and four phytocassanes (e.g. phytocassane D, 18) (Koga et al., 1995). Many genes involved in rice resistance to the blast fungus have been identi®ed, and it is very likely that at least some of these genes code for a quick response by the plant's defence system and the production of high concentrations of phytoalexins. Some of the criteria proposed by Subba Rao and Strange (1994) cited above were tested to see whether phytoalexins play a role in the resistance of rice plants to the blast pathogen, using the highly resistant rice cultivar Tetep. Leaves were punch-inoculated with an avirulent strain of Pyricularia oryzae. After ®xed periods of 3±5.8 days the concentrations of the rice phytoalexins sakuranetin, oryzalexins D, E and S, and momilactone A were determined, because standard compounds were available for these (Dillon et al., 1997). Three days after inoculation, the ®rst three criteria were met by at least two of the phytoalexins produced, sakuranetin and momilactone A. To see whether higher quantities of phytoalexins are accumulated by rice genotypes with a high degree of resistance than by genotypes with a low resistance (criterion 4), a number of rice genotypes of dierent susceptibility
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to Pyricularia oryzae were tested. These were obtained from the International Rice Research Institute, The Philippines, and from the Central Rice Breeding Station, Sri Lanka. Phytoalexins were induced by means of shortwave UV irradiation of the leaves, because this method was quick and gave the most consistent results between replicates (Grayer et al., 1996). The experiments showed that there are signi®cant and consistent dierences among rice cultivars in their phytoalexin response to UV irradiation. The phytoalexins that have the highest antifungal activity (sakuranetin, momilactone A and oryzalexin S) were produced in very much higher quantities in the resistant cultivars Tetep and Ta-poo-cho-z than in the susceptible IR 50 and B 40. Indeed, B 40 did not produce any sakuranetin or oryzalexin S (Dillon et al., 1997). Of course, the phytoalexin response of rice plants to a fungus may be completely dierent from their response to UV irradiation. However, although the results of phytoalexin production by Tetep after inoculation with P. oryzae and after UV irradiation were not completely identical, they showed a similar trend. These results, and those obtained for resistant genotype Ta-poocho-z and for susceptible IR 50 and B 40 after UV irradiation, strongly suggest that phytoalexin production could be
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an important factor in the resistance of rice against the rice blast pathogen. The resistant Carreon, however, failed to produce high concentrations of phytoalexins. This cultivar showed hardly any necrosis after UV irradiation, in contrast to the other four cultivars. This may explain its low phytoalexin production. It is possible that infection with a pathogen is a more eective way of inducing phytoalexins in Carreon, but it is also possible that blast resistance in Carreon is based on dierent factors, e.g. a very impenetrable epidermis or multifunctional exudate chemicals possessing both antimicrobial activity and UV-®ltering functions. This would be worth investigating further. The results obtained with Carreon mean that some resistant rice genotypes may be overlooked in a screening programme based phytoalexin production alone, even if a pathogen is used for elicitation rather than an abiotic elicitor. Furthermore, if an abiotic elicitor such as UV-irradiation is employed, there is no guarantee that this always will result in the induction of phytoalexins, even if a biotic elicitor would have done so. Nevertheless, a biochemical approach could complement ®eld trials and oer a number of advantages. 8. Prospects of phytoalexin research Because induction of phytoalexins is now recognised as an important mechanism of plants to defend themselves against fungal pathogens, plant pathologists should be able to exploit this ability of plants to combat plant diseases. When phytoalexin research was in its infancy, there were great expectations that phytoalexins could be utilised as novel natural fungicides. So far this prospect has proved to be rather disappointing. The toxicity of many phytoalexins appears to be rather unspeci®c, so that they often are as toxic to other organisms including humans as they are to fungi. Furthermore, their fungicidal activity is generally much lower than that of commercial fungicides already on the market, and they are generally very expensive to isolate or synthesise (KucÂ, 1991). More highly active analogues may be produced, but these have the same environmental disadvantages as synthetic fungicides currently in use. As mentioned before, phytoalexins are not only induced or elicited in plants by microorganisms, but also by UV irradiation and a range of chemicals, including heavy metal salts and certain cell wall components of fungi including chitin (all agents that can induce phytoalexins are called `elicitors'). Therefore, an alternative to protect crop plants would be to spray them with environmentally harmless phytoalexin elicitors. Sinha (1994) tested a large variety of dierent elicitors to see what their eect was on the prevention of various fungal diseases in a number of crop plants and achieved good results. However, according to Kuc (1991), the repeated application of elicitors of phytoalexin accu-
mulation can cause a marked diversion of energy and carbon resources from vital processes, resulting in resistant but stunted plants. Therefore crop yields may be less than if the plants were infected! Plant resistance can also be enhanced by incorporation of genes coding for phytoalexin synthesis. For instance, the gene for stilbene synthase has been successfully transferred from the grape vine, Vitis vinifera, to tobacco plants, so that the latter produced the stilbene phytoalexin resveratrol, and acquired improved resistance to infection by Botrytis cinerea (Hain et al., 1993). However, it is relatively easy to modify plants genetically to produce resveratrol, as this only needs one enzyme (the other enzymes needed for its biosynthesis are present in most plants). It is not so easy to do the same for many other phytoalexins. For instance, for non-leguminous plants many new enzymes would be required to produce pterocarpan phytoalexins, so that many genes would have to be transferred. Furthermore, the enhancement of plant resistance by gene transfer has several undesirable aspects. If the genes coding for the production of a particular powerful phytoalexin were to be transferred to many crop plants, pathogens could develop that could break that resistance factor, e.g. by evolving enzymes to detoxify that particular phytoalexin. These could become `superbugs' and wipe out not only all the genetically modi®ed crops, but also any wild plant species that were dependent on that phytoalexin for their defence. The transfer of genes to produce a particular phytoalexin may not render a plant resistant to diseases, because it is not usually the inability of a plant to produce these compounds that makes it susceptible. The speed and magnitude of the gene expression, i.e. to produce the phytoalexins rapidly and in high enough concentrations, may be a more important factor (KucÂ, 1991). For instance, the susceptible rice genotype B 40 does not lack the ability to synthesise phytoalexins, but produces them in much lower concentrations than the resistant genotypes Tetep and Ta-poo-cho-z (Dillon et al., 1997). Therefore, a better way of utilising the knowledge of phytoalexin production in plants for crop protection is to select genotypes for their yield of induced antifungal compounds and their speed of phytoalexin response. Another way would be to stimulate the plants (even susceptible genotypes) to produce their own phytoalexins rapidly and in large quantities. This can be done by means of immunisation, i.e. by inoculation with a non-pathogenic microorganism or an incompatible strain of a pathogen. This causes a systemic resistance: after any subsequent infection with a pathogen the plant responds rapidly to accumulate phytoalexins at the site of infection and produces lignin and other products to halt the growth of the pathogen (KucÂ, 1991). For example, systemic resistance to Pyricularia oryzae in rice was induced by inoculation of the ®rst leaf with the bacterium Pseudomonas syringae pv. syringae, which
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causes a hypersensitive response in rice. Lesions caused by a subsequent inoculation with Pyricularia oryzae were dramatically decreased in number and size (Smith and MeÂtraux, 1991). This resistance may have been the result of a rapid production of phytoalexins, although this was not investigated. Apart from having various dierent potentials for agriculture, phytoalexin production is also a treasure chest for other ®elds of science, including natural product chemistry, pharmacy, ecology, biochemistry and molecular biology. For the chemist phytoalexins present ample opportunities for elucidating novel structures. Because these compounds are not usually present in healthy plants, they are not often found during standard plant screening programmes. Moreover, only some 40 out of the ca. 150 plant families of Angiospermae and Gymnospermae have been surveyed yet for their phytoalexin response (Harborne, 1999), so many new discoveries could still be made. For the pharmacist phytoalexins have the potential to show pharmacological activities, as these compounds are by de®nition antimicrobial. Interesting areas of research for biochemists are the biosynthesis and enzymes involved in the production of the myriad of phytoalexins found so far. The de novo synthesis of phytoalexins provides molecular biologists with a very convenient source for studying gene regulation mechanisms, because new expressions of genes take place in a very short time. This is a rapidly expanding area of phytoalexin research at the moment. Finally, the plant's strategy of producing phytoalexins is a worthwhile object of study for ecologists, because this has contributed to the survival of all extant plant species on which all other organisms ultimately depend. Acknowledgements We thank the publishing editor of Phytochemistry for inviting us to present this paper and members of the Plant Science Laboratories, University of Reading, for their support and continuous encouragement throughout the projects.
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ReneÂe Grayer studied biology with chemistry at the University of Leiden, The Netherlands. She attained a PhD at the same university on the chemotaxonomy of the genus Veronica (Scrophulariaceae), supervised by Prof. Robert Hegnauer. In 1971 she spent 3 months in Prof. Jerey Harborne's phytochemical unit at the University of Reading to learn techniques to separate and identify ¯avonoids, and subsequently she worked in that laboratory from 1976 to 1994 on a variety of projects. These included the chemotaxonomy of the Leguminosae, ¯avonoid sweeteners from plants, and the chemical basis of resistance of crop plants to insect
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pests and fungal diseases. In 1994 she took up a position at the Royal Botanic Gardens, Kew (Biological Interactions Section, Jodrell Laboratory), where she mainly works on the chemotaxonomy of the Lamiaceae. Together with Jerey she wrote a number of research papers, review articles and chapters of books, and she also wrote many entries for the Phytochemical Dictionary and Handbook of Natural Flavonoids, both edited by Jerey Harborne and Herbert Baxter. Since 1987 she has compiled the Index to the journal Phytochemistry, taking over from Tony Swain.
Tetsuo Kokubun left his native Japan in the summer of 1991 to do a PhD with Jerey Harborne at the University of Reading, UK. The aim of his study was to ®nd phytoalexins in the Rosaceae. The Botanical Garden at the university supplied the living plant material for this study, and Jerey's enthusiasm the inspiration. This work resulted in seven papers, mostly published in this journal. In 1997, after postdoctoral positions in Auckland, New Zealand, and in Reading, he moved to the Biological Interactions Section, Jodrell Laboratory, Royal Botanic Gardens, Kew, where he now works on bioactive fungal metabolites and antimicrobial compounds from higher plants. However, he has always stayed in touch with Jerey, and recently assisted him in the compilation and editing of the Handbook of Natural Flavonoids Ð the latest compendium in the ®eld.