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SUMMARY

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

Alternaria species are mainly saprophytic fungi. However, some species have acquired pathogenic capacities collectively causing disease over a broad host range. This review summarizes the knowledge on pathogenic strategies employed by the fungus to plunder the host. Furthermore, strategies employed by potential host plants in order to ward off an attack are discussed.

Taxonomy:Alternaria spp. kingdom Fungi, subkingdom Eumycotera, phylum Fungi Imperfecti (a non-phylogenetic or artificial phylum of fungi without known sexual stages whose members may or may not be related; taxonomy does not reflect relationships), form class Hypomycetes, Form order Moniliales, form family Dematiaceae, genus Alternaria. Some species of Alternaria are the asexual anamorph of the ascomycete Pleospora while others are speculated to be anamorphs of Leptosphaeria.

Host Range: Most Alternaria species are common saprophytes that derive energy as a result of cellulytic activity and are found in a variety of habitats as ubiquitous agents of decay. Some species are plant pathogens that cause a range of economically important diseases like stem cancer, leaf blight or leaf spot on a large variety of crops. Latent infections can occur and result in post-harvest diseases or damping-off in case of infected seed.


INTRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

Most Alternaria species are saprophytes that are commonly found in soil or on decaying plant tissues. Some species are (opportunistic) plant pathogens that, collectively, cause a range of diseases with economic impact on a large variety of important agronomic host plants including cereals, ornamentals, oilcrops, vegetables such as cauliflower, broccoli, carrot and potato, and fruits like tomato, citrus and apple. Alternaria spp. are also well known as post-harvest pathogens. Some Alternaria spp. are of clinical significance as they are well known for the production of toxic secondary metabolites, some of which are powerful mycotoxins that have been implicated in the development of cancer in mammals. A. alternata in particular is gaining prominence as an emerging human pathogen, especially in immuno-compromised patients. In addition, Alternaria spores are one of the most common airborne allergens.

The two major features of Alternaria species are the production of melanin, especially in the spores, and the production of host-specific toxins in the case of pathogenic species. Both features will be discussed in this review. Furthermore, this review discusses the pathogenic strategies employed by Alternaria spp. to parasitize host plants and defensive reactions employed by the host to stop pathogen ingress.

CLASSIFICATION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

The genus Alternaria was established in 1817 with A. alternata (originally A. tenuis) as the type isolate. Because of the absence of an identified sexual stage for the vast majority of Alternaria species, this genus was classified into the division of mitosporic fungi or the phylum Fungi Imperfecti. The key taxonomic feature of the genus Alternaria is the production of large, multicellular, dark-coloured (melanized) conidia with longitudinal as well as transverse septa (phaeodictyospores). These conidia are broadest near the base and gradually taper to an elongated beak, providing a club-like appearance (Fig. 1A,G). They are produced in single or branched chains on short, erect conidiophores. Alternaria forms conidia that arise as protrusions of the protoplast through pores in the conidiophore cell wall. At the onset of conidial development, the apex of the conidiophore thickens and a ring-shaped electron-transparent structure is deposited at the apical dome. At the central cavity of this electron-transparent structure a pore is formed through the dissolving of the cell wall. Through this pore, cytoplasm only covered by the plasma membrane is pushed out. The turgor pressure required to push the cytoplasm through the pore is presumably provided by the well-developed, large vacuole that appears at this stage in the conidiophore cell. Subsequent to this, a nucleus migrates into the new-borne conidium and later on a cell wall is deposited (Honda et al., 1987, 1990). The melanin that is present in the conidia is concentrated in the outer region of the primary cell walls, which are derived from the original wall of the developing spore, and in the septa, which delimit individual spore cells in the multicellular conidium. After the cells have been delimited by septa, secondary cell walls are deposited, but these remain unmelanized, suggesting a developmental regulation of melanin deposition during conidiogenesis (Campbell, 1969; Carzaniga et al., 2002; Kawamura et al., 1997). Melanin is probably actively involved in conidial development, since disruption of a melanin biosynthesis gene in A. alternata reduced conidial size as well as septal number (Kawamura et al., 1999).

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Figure 1. Symptoms caused by, and appearance of Alternaria spp. (A) Stand of A. alternata conidiophores with chains of conidia (picture kindly provided by G. Barron). (B) Germinating conidia of A. alternata f.sp. citri, the causal agent of brown spot, infecting a citrus leaf (SEM picture kindly provided by A. Bhatia and P. Timmer). (C) Black spot on potato caused by A. solani (picture kindly provided by Carlos A. Lopes). (D) Typical ‘target spot’ symptom of Alternaria: a series of concentric rings at the site of attack. (E) A. brassicicola on a susceptible Arabidopsis leaf. (F) developing chains of A. brassicicola conidia on the surface of an inoculated Arabidopsis leaf. (G) A. brassicicola conidia with longitudinal as well as transverse septa (phaeodictyospores).

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A classification based on conidial characteristics is complicated by the existence of other fungal genera, such as Stemphylium and Ulocladium, which produce phaeodictyosporic conidia that resemble those of Alternaria. Based on the characteristics defined by Simmons (1995), Stemphylium and Alternaria species are discriminated by the appearance of the conidiophore apex, and Ulocladium and Alternaria species by the appearance of the basal end of immature conidia. This differentiation is largely supported by a molecular analysis of ribosomal DNA sequences (Pryor and Gilbertson, 2000).

Within the genus Alternaria, species are also primarily defined upon conidium characteristics. Over 100 species occurring world-wide have been described (Simmons, 1992). However, errors in the taxonomy of Alternaria species have arisen due to the variability of its morphological characters, which are not only affected by intrinsic factors but also by environmental conditions. As a result of this, it is feasible that single species have been accidentally divided into several (Rotem, 1994). This is illustrated by the observation that the species A. alternata alone is capable of attacking over 100 hosts and, in addition, Groves and Skolko (1944) found that this species typically displays morphological variations. Obviously, this phenotypic variation does not justify assigning A. alternata-like specimens to other species. However, it is this morphological variation that has probably resulted the description of certain Alternaria species that have never been verified by others (Rotem, 1994).

Because of the large diversity of Alternaria species, a division into subgeneric groups has occasionally been proposed. However, to date there has not been one general classification of Alternaria. Neergaard (1945) proposed a classification based on catenulation (chain-formation of conidia; Fig. 1A,F), while more recently an organization of the genus into species groups, each typified by a representative species, was proposed (Simmons, 1992). Because of morphological similarity, but pathological differences, strains of a certain species, especially A. alternata, have been defined as formae speciales or ‘pathotypes’ (Nishimura and Kohmoto, 1983).

MELANIN PRODUCTION

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

Melanins are dark, brown to black, high molecular weight pigments that are produced by organisms ranging from animals and plants to micro-organisms. They are formed by the oxidative polymerization of phenolic or indolic compounds. Because polymerization does not follow an exact pattern, melanins are molecules with various structures carrying aromatic rings and available hydroxyl groups (Bell and Wheeler, 1986). Remarkably, melanin contains stable populations of free radicals (Enochs et al., 1993). Some fungi produce melanin through the oxidation of environmentally obtained l-3,4-dihydroxyphenylalanine (DOPA; Butler and Day, 1998). However, most fungal melanins, including those produced by Alternaria, appear to be derived from the monomeric precursor 1,8-dihydroxynaphthalene (DHN) which is produced through the pentaketide pathway (Kimura and Tsuge, 1993; Fig. 2). It is thought that DHN is produced in the cytoplasm and subsequently exported into the cell wall and extracellular matrix, where laccases catalyse the final polymerization to melanin (Bell and Wheeler, 1986; Butler and Day, 1998).

image

Figure 2. Pentaketide pathway leading to the formation of DHN from acetate in A. alternata and other fungi. Through condensation and cyclization, 1,3,6,8-tetrahydroxynaphthalene is formed from acetate. Through alternating reduction and dehydration, 1,8-dihydroxynaphthalene (DHN) is formed which can be directly formed into melanin through oxidative polymerization.

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Apart from a role in conidial development (Kawamura et al., 1999), melanins appear to have an indirect as well as a direct function in virulence. On the one hand they act as ‘body armour’, protecting fungi against environmental stress or unfavourable conditions like extreme temperatures, UV-radiation and compounds secreted by microbial antagonists, thus adding to longevity and survival (Kawamura et al., 1999; Lockwood, 1960; Rehnstrom and Free, 1996). Indeed, for Cochliobolus heterostrophus, the cause of Southern corn leaf blight, it was demonstrated that, although virulence under laboratory conditions was sustained, melanin-deficient isolates lost their capacity to cause disease in the field (Frye et al., 1984).

On the other hand, for some fungi a direct function of melanin in virulence has also been demonstrated. Magnaporthe and Colletotrichum species produce non-melanized spores that, after germination, lead to the formation of well-developed melanized appressoria. For these fungi it has been demonstrated that the melanization of the appressorium contributes to fungal virulence, as mutants that no longer accumulate melanin do not form these well-developed appressoria and are incapable of penetrating the host (Howard and Valent, 1996). In addition, melanin not only appears to contribute to virulence through the functioning of the appressorium, but can also act as a scavenger of free oxygen radicals, which can be components of host defence against pathogen ingress (Jacobson et al., 1995; Wang and Casadevall, 1994). Finally, synthetic melanin has been found to have immunosuppressive characteristics in vitro on mammalian cells (Mohagheghpour et al., 2000). Although a similar action in vivo has not been demonstrated yet, it is tempting to speculate that similar activities could also be displayed in plant infections.

There is a clear difference in the requirement of melanin for pathogenicity between fungal species, explaining the diversity in timing, localization and function of melanin synthesis. Despite this diversity, the production of melanin through the pentaketide pathway seems to be conserved among fungi, including Alternaria. The gene cluster required for melanin synthesis was cloned from A. alternata (Kimura and Tsuge, 1993). Alternaria naturally produces unmelanized appressoria and A. alternata mutants that are deficient in melanin production retain their pathogenicity, demonstrating that melanin is not required for its virulence (Tanabe et al., 1995). Despite the fact that Alternaria produces unmelanized appressoria, an Alternaria gene encoding a polyketide synthase can complement the appressorial melanization of a Colletotrichum lagenarium albino mutant (Takano et al., 1997). However, the cell wall architecture of complemented C. lagenarium appressoria differed from wild-type appressoria and complemented appressoria showed restored penetration of cellulose membranes but not of cucumber cotelydons (Takano et al., 1997). Such differences are likely to be caused by differences in pentaketide-pathway gene regulation. This was indeed demonstrated in Colletotrichum and Magnaporthe, for which transcription factors were identified that are involved in the developmental regulation of melanin synthesis (Tsuji et al., 2000).

THE INFECTION PROCESS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

In general, Alternaria species are foliar pathogens that cause a relatively slow destruction of host tissues through the reduction of photosynthetic potential. An infection leads to the formation of necrotic lesions (Fig. 1C–E), which sometimes have a target-like appearance due to growth interruptions caused by unfavourable conditions (Fig. 1D). The fungus resides in the centre of the lesion, which is surrounded by an un-invaded chlorotic halo, a symptom that is commonly observed for the infection process of necrotrophic pathogens. This zone is created by the diffusion of fungal metabolites like toxins (Agarwal et al., 1997; Tewari, 1983). Members of the genus Alternaria frequently cause quiescent infections in which the fungus enters the tissue where it remains dormant until changed conditions favour infection. Alternaria species generally do not affect water or nutrient transport throughout the plant, because they do not specifically target roots or vessels (Rotem, 1994).

Alternaria has no known sexual stage or overwintering spores, but the fungus can survive as mycelium or spores on decaying plant debris for a considerable time, or as a latent infection in seeds (Rotem, 1994). If seed-borne, the fungus can attack the seedling once the seed has germinated. In other cases, once the spores are produced they are mainly spread by wind on to plant surfaces where infection can occur. Typically, weakened tissues, either due to stresses, senescence or wounding, are more susceptible to Alternaria infection than healthy tissues. The observation that saprobic Alternaria species can become parasitic when they meet a weakened host illustrates that the distinction between saprophytic and parasitic behaviour is not always evident.

Despite the taxonomic and pathogenic differences between Alternaria species, they cause similar infection patterns. Dormant spores have heavily melanized walls that, under favourable conditions, produce one or more germ tubes (Fig. 1B). Subsequently, the germ tubes penetrate stomata, cuticle or wounds with or without the formation of small appressoria. In less virulent species, wounds and stomata are targeted, while more virulent species can also penetrate directly (Rotem, 1994). Enzymatic processes in Alternaria infections are essentially similar to those in other diseases. The cuticle, which consists of a combination of cutin (a hydroxyl fatty acid polyester) and waxes, comprises the first line of defence to be overcome by directly penetrating fungal pathogens. For A. brassicicola, the differential expression of cutinase genes was monitored between saprophytic and pathogenic stages of the fungus (Yao and Köller, 1995). Furthermore, it was found that different cutinolytic enzymes are sequentially induced upon landing on, and penetration of, the cabbage leaf (Fan and Köller, 1998). Constitutively produced cutinases are expressed during the initial contact of A. brassicicola with the cuticle. After reaching subcuticular layers, different cutinases that are active during saprophytic growth are induced (Trail and Köller, 1993; Yao and Köller, 1994). These cutinases are inducible by cutin monomers. This implies a switch between the parasitic and the saprophytic stage of the fungal pathogen. So far, however, it has not been demonstrated that any extracellular hydrolase is crucially involved in fungal pathogenesis.

In addition to cutinases, lipases might also contribute to the establishment of infection. A. brassicicola was found to produce a lipase that acts as a virulence factor (Berto et al., 1997). Anti-lipase antibodies were found to have an inhibitory effect on the in vivo infection of cauliflower leaves, as symptom development was inhibited in a dose-dependent manner despite normal germination of spores. However, the antibodies did not have an effect on fungal infection of dewaxed leaves, which suggests that this lipase has an early pathogenic activity during penetration (Berto et al., 1999).

About one-third of the total cell wall components in dicotyledonous plants are pectic polysaccharides. These components can be hydrolized by fungal galacturonidases. A. citri was found to be dependent on endopolygalacturonidase activity for establishing an infection, as a mutant lacking this activity was severely compromised in its pathogenic capacity. On the other hand, the tangerine pathotype of A. alternata did not show this dependency, possibly because this particular pathogen largely depends on toxin production for the colonization of its host (Isshiki et al., 2001).

For a specific A. alternata endoglucanase it was demonstrated that its production is triggered by a pathogen-induced pH increase on the host (Eshel et al., 2002b). Correlation-studies between enzyme production and symptom development suggest that endoglucanases, and also exo-glucanases, are involved in A. alternata pathogenicity (Eshel et al., 2000, 2002a,b).

TOXIN BIOSYNTHESIS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

Most phytotoxins employed by a fungal invader are chemically diverse secondary metabolites: low molecular weight components that are not required for normal growth or reproduction. Often these toxins are produced as families of related compounds. Based on selectivity, phytotoxins can be divided into two categories: non-host-specific toxins and host-specific toxins. In general, non-host-specific toxins have relatively mild phytotoxic effects, affect a broad spectrum of plant species and are thought to be an additional factor of disease alongside, for instance, penetration mechanisms and enzymatic processes. Although they generally act as virulence factors and intensify disease symptom severity, they are not absolutely required for establishing disease since they are also toxic to plant species outside the host range of the pathogen. They merely precondition the host for disease (Ballio, 1991).

In Alternaria, many non-host-specific toxins have been identified, although the precise action of only a few has been studied in detail. Brefeldin A (dehydro-)curvularin, tenuazonic acid, tentoxin and zinniol are examples of toxins that are produced by several Alternaria species. They exert their phytotoxic activity through different modes. Brefeldin A causes disassembly of the Golgi complex and acts as an inhibitor of secretion, while curvularin is an inhibitor of cell division through its disturbance of the microtubule assembly, tenuazonic acid inhibits protein synthesis and zinniol affects membrane permeabilization (Fujiwara et al., 1988; Meronuck et al., 1972; Robeson and Strobel, 1981; Thuleau et al., 1988). Tentoxin is produced by A. alternata and acts as a photophosphorylation inhibitor through specific binding to chloroplast ATP synthase, causing the inhibition of ATP hydrolysis and ATP synthesis (Steele et al., 1978). Because these toxins often target basic cellular processes, they are often powerful mycotoxins.

Host-specific toxins are involved in the development of a few, destructive diseases. They generally display severe effects on a rather narrow species-range that serves as host to the fungus and they are indispensable for disease. Along with species of the genus Cochliobolus, Alternaria species are known to produce host-specific toxins (reviewed in Markham and Hille, 2001; Wolpert et al., 2002). There are at least 12 host-specific toxin-producing plant pathogenic Alternaria species, most of which appear to be variants of A. alternata (Fig. 3). It was proposed that these variants should be considered pathotypes of A. alternata (Nishimura and Kohmoto, 1983), a hypothesis that is supported by molecular analysis (Kusaba and Tsuge, 1994, 1995, 1997). The toxins that are produced by these pathotypes are chemically diverse, ranging from low molecular weight secondary metabolites to peptides (Fig. 3). In a study on pathogenic (host-specific toxin-producing) and non-pathogenic (non-host-specific toxin-producing) A. alternata species, it was established that all pathotypes tested carried (small) extra chromosomes, whereas the non-pathogenic ones did not carry these extra chromosomes (Akamatsu et al., 1999). In fungal species, one can regularly find extra chromosomes in certain subsets of individuals, so-called supernumerary chromosomes. These chromosomes are not essential because they are not required for normal growth, but they can carry extra traits. If supernumerary chromosomes confer an adaptive advantage to the individual in some habitats, they are referred to as conditionally dispensable chromosomes. The production of the cyclic AM-toxin peptide by the apple pathogen A. alternata f.sp. mali is determined by such a chromosome (Johnson et al., 2000, 2001). The AK- and AF-toxin biosynthesis genes and their homologues, identified in the Japanese pear and strawberry pathotypes of A. alternata, respectively, generally clustered together on a small chromosome (Hatta et al., 2002; Tanaka and Tsuge, 2000; Tanaka et al., 1999). Physical clustering is a phenomenon that is commonly found for genes involved in the production of secondary metabolites in fungi (Keller and Hohn, 1997).

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Figure 3. Chemical structures of Alternaria host-specific toxins. Most of these toxins are produced as families of related compounds, of which only the major compound is shown. The chemical structures of AT-toxin produced by the tobacco pathotype of A. alternata (previously A. longipes), ATC-toxin produced by the pigeon pea pathogen A. tenuissima and AB-toxin produced by A. brassicicola are currently not known (Kodama et al., 1990; Nutsugah et al., 1994; Otani et al., 1998).

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AK- and AF-toxin are related to ACT-toxin, produced by the tangerine pathotype of A. alternata, and carry an epoxy-decatrienoic ester backbone (Fig. 3). Interestingly, the AF- and ACT-producing strains were also found to be pathogenic on pear lines that are sensitive to AK-toxin, but not vice versa (Kohmoto et al., 1993; Maekawa et al., 1984). Four genes involved in the biosynthesis of AK-toxin have been cloned from the Japanese pear pathotype genome of A. alternata, and homologues of three of these genes have also been identified in the strawberry and tangerine pathotypes. This suggests that these homologues are involved in the production of common moieties between these toxins (Hatta et al., 2002; Masunaka et al., 2000; Tanaka and Tsuge, 2000; Tanaka et al., 1999). Together with the existence of such homologous toxin biosynthesis genes between pathotypes, the physical clustering of toxin genes on a single chromosome suggests that these genes are acquired through horizontal gene transfer (Tanaka et al., 1999; Walton, 2000). This hypothesis is supported by the observation that homologues of toxin biosynthesis genes have not been found in strains from other pathotypes or non-pathogenic A. alternata isolates, making it unlikely that mutations or internal genetic rearrangements account for the toxin-producing ability (Hatta et al., 2002; Masunaka et al., 2000; Tanaka et al., 1999). There is no advantage for the clustering of genes during gene transmission from one generation to the next (vertical gene transfer), because the entire genome is transferred as a unit. However, during horizontal gene transfer, a relatively small contiguous DNA sequence is generally transferred. Since the total set of biosynthesis genes should be transmitted from one species to the other in order to provide the recipient with a complete biosynthesis pathway, this chance is higher if genes are clustered. An originally saprophytic fungus like Alternaria could have acquired pathogenic capacity in this way (Walton, 2000), an hypothesis which is supported by the finding that single pathotype populations do not form monophyletic groups (Kusaba and Tsuge, 1994, 1995, 1997). The occurrence of horizontal gene transfer is well accepted for prokaryotes and even for fungal mitochondrial genes, but experimental evidence for the horizontal gene transfer of fungal nuclear genes is scarce. However, the difference between patterns of repeated DNA sequences on certain supernumerary chromosomes and the rest of the chromosomes in the same genome suggest that both types have a different evolutionary history (Covert, 1998). Furthermore, it was demonstrated for the fungus Colletotrichum gloeosporioides that a small chromosome could be transferred between two genetically isolated strains, thus demonstrating that horizontal gene transfer can occur (He et al., 1998).

MODE OF ACTION OF HOST-SPECIFIC TOXINS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

Although the site of action of different Alternaria toxins varies, in the end they all trigger host cell death. AF-, ACT- and ACTG-toxin act at the plasma membrane and cause permeabilization (Otani et al., 1995). AM-toxin not only affects the plasma membrane, but also acts on chloroplasts, while ACT- and AT-toxin were found to affect mitochondria (Otani et al., 1995). ACR-toxin induces swelling and other morphological modifications of mitochondria, and increases NADH oxidation, which is followed by plasma membrane disorders leading to electrolyte leakage and necrosis (Akimitsu et al., 1989). For most of these toxins, however, their mechanism of action is barely understood.

AAL-toxin is an aminopentol ester, an analogue of the sphingosine precursor sphinganine, which is produced by the tomato pathogen A. alternata f.sp. lycopersici (Fig. 3). This toxin closely resembles the mycotoxin Fumonisin B1 that was identified as a Fusarium moniliforme toxin, and is also produced by AAL-toxin producing A. alternata species (Chen et al., 1992). Interestingly, Fumonisin B1 is also selectively toxic to AAL-toxin sensitive tomato genotypes (Gilchrist et al., 1992). A. alternata mutants affected in AAL-toxin production also lose their pathogenicity, demonstrating the requirement of this toxin for pathogenicity (Akamatsu et al., 1997). Like Fumonisin B1, AAL-toxin is an inhibitor of sphingolipid (ceramide) biosynthesis (Abbas et al., 1994; Spassieva et al., 2002). The application of AAL-toxin leads to an accumulation of sphingoid base precursors, a depletion of complex sphingolipids and subsequently to the cell death of sensitive tomato species (Abbas et al., 1994; Wang et al., 1996b). Similar effects of the toxin are observed on mammalian cells (Wang et al., 1996a). In eukaryotic cells, ceramides modulate a number of biochemical and cellular stress responses such as cell-cycle arrest and apoptosis (Hannun, 1996). The AAL-toxin induced cell death observed in plant cells shares characteristics with genetically controlled cellular suicide, or apoptosis, as observed in animals, thus suggesting an active participation of the host in AAL-toxin mediated cell death (Wang et al., 1996b). Indeed, transgenic tomato plants expressing a viral anti-apoptotic gene were protected against AAL-toxin-induced cell death and pathogen infection (Lincoln et al., 2002). Use of the host's suicide programme as a compatibility factor was not only shown for AAL-toxin producing A. alternata strains, but seems to be a general strategy of necrotrophic pathogens (Dickman et al., 2001; Lincoln et al., 2002).

In plants, insensitivity to AAL-toxin and resistance to AAL-producing A. alternata f.sp. lycopersici is determined by the co-dominant Alternaria stem cancer (Asc-1) gene, a homologue of a yeast longevity assurance gene of which homologues are also found in nematodes and humans (Brandwagt et al., 2000, 2002; Spassieva et al., 2002). Although the precise mode of action of Asc-1 has not yet been determined, based on studies with human and nematode homologues in yeast it is believed that ASC1 relieves a toxin-induced block on sphingolipid synthesis through salvage of the ER-to-Golgi transport of GPI-anchored proteins (Brandwagt et al., 2000).

In addition to a poor knowledge of the mechanism of action of different host-specific toxins, the basis of host-specificity is only understood for a few of them. For destruxin B, a host-selective phytotoxin produced by Alternaria brassicae (Fig. 3; Pedras et al., 2002) the mechanism for host-selectivity was recently revealed. The selective phytotoxicity appears to be due to a fast and efficient detoxification, by sequential hydroxylation and glycosylation reactions, in tissues of resistant species. These reactions were also found to occur in susceptible species, but at a slower rate, providing an explanation for selective toxicity (Pedras et al., 2001). Similarly, toxin detoxification has been demonstrated to determine resistance against the host-specific toxin producing fungus Cochliobolus carbonum (Multani et al., 1998).

For ACR-toxin, specificity seems to be determined by differential post-transcriptional processing of a mitochondrial gene. This gene is present in the mitochondrial DNA of toxin-sensitive as well as resistant species, but the transcript of the gene is shorter in resistant than in sensitive mitochondria. In the end, an oligomeric protein is produced in toxin-sensitive mitochondria, whereas the transcript is not translated in resistant mitochondria. Although the gene is likely to encode a mitochondrial membrane protein, no function has been assigned yet (Ohtani et al., 2002).

HOST DEFENCE AGAINST ALTERNARIA SPECIES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

As mentioned before, destruxin B is a host-specific Alternaria phytotoxin whose selective phytotoxicity is due to the detoxification in tissues of resistant species (Pedras et al., 2001). In addition, the hydroxylation product that is generated during detoxification was found to induce phytoalexin biosynthesis in resistant, but not in susceptible, plant species. This suggests that resistance depends on phytotoxin detoxification and simultaneous phytoalexin elicitation (Pedras et al., 2001). Phytoalexins have also been implicated in pathogen containment for other Alternaria–host interactions. In Camelina sativa (false flax), resistance against A. brassicae has been correlated with production of the phytoalexin camalexin (Jejelowo et al., 1991). Moreover, Arabidopsis synthesizes camalexin upon pathogen challenge. Since Arabidopsis is amendable to phytopathological studies as well as molecular genetics, it has been established as a model for dissecting plant defence mechanisms. The Arabidopsis pad3 mutant (phytoalexin deficient) was found to be deficient in camalexin biosynthesis and to display severely enhanced susceptibility to Alternaria brassicicola, demonstrating that camalexin contributes to Alternaria resistance (Thomma et al., 1999b). Although camalexin was found to exhibit direct antimicrobial activity to this fungus in vitro (Thomma et al., 1999b), it cannot be excluded that camalexin also contributes to Alternaria resistance in an indirect way, as camalexin was found to inhibit production of the host-specific toxin destruxin B in A. brassicae (Pedras et al., 1998).

In a study of 24 different Arabidopsis ecotypes it was not possible to discover a direct relationship between camalexin production upon A. brassicicola inoculation on one hand, and resistance against this pathogen on the other (Kagan and Hammerschmidt, 2002). This is perhaps not surprising since defence against A. brassicicola in Arabidopsis appears to depend on multiple defence components, the relative importance of which can vary between ecotypes. Previously, jasmonate has also been implicated in defence against Alternaria, as the jasmonate-insensitive Arabidopsis mutant coi1 was found to display enhanced susceptibility to this pathogen (Thomma et al., 1998, 1999a, 2000). Jasmonate is a plant hormone that, along with salicylic acid and ethylene, plays a role in the activation of defence responses in plants (Thomma et al., 2001). However, mutants affected in either salicylic acid or ethylene signalling are as resistant as wild-type plants, leading to the conclusion that neither salicylic acid nor ethylene is directly involved in resistance against Alternaria in Arabidopsis (Thomma et al., 1998, 1999a). Jasmonate was found not to directly induce camalexin biosynthesis and, in addition, jasmonate-insensitivity does not lead to camalexin deficiency. It was therefore concluded that both camalexin and jasmonate mediate host resistance against Alternaria through separate but parallel pathways in Arabidopsis (Thomma et al., 1998, 1999b, 2001). Further substantiation for this observation came from the finding that the Arabidopsis mutation esa1 affects resistance against A. brassicicola through a severe reduction in both camalexin-production as well jasmonate-dependent gene induction (Tierens et al., 2002). It is currently not clear, however, which jasmonate-inducible effector molecules are responsible for containment of Alternaria in Arabidopsis.

In a cDNA microarray analysis on a set of 2375 Arabidopsis genes, 168 genes were found to be up-regulated and 39 were found to be repressed 72 h after inoculation with A. brassicicola. About 10% of these 207 genes can be implicated in plant defence and about one-third in cell maintenance and development (Schenk et al., 2000). However, a precise role for any of the responsive genes in the defence of Arabidopsis against A. brassicicola has not yet been established. From studies using ethylene- and jasmonate-insensitive Arabidopsis mutants it can be concluded that a set of PR-genes encoding plant defensin, hevein-like protein and basic chitinase are not markedly involved in Alternaria resistance (Thomma et al., 1998, 1999a). For tomato, PR-proteins have been implicated in Alternaria resistance through the observation that resistance in tomato against A. solani correlates with high and rapid accumulation of PR-proteins. However, it is speculated that those PR-proteins, many of which possess hydrolytic activity, are not necessarily directly involved in arresting the pathogen but rather release elicitors from the pathogen cell wall, thus triggering a HR (Lawrence et al., 2000).

The production of reactive oxygen species is one of the earliest defence reactions of plants against pathogen attack. In the presence of tobacco plant extracts, A. alternata was found to synthesize mannitol, which can act as a potent quencher of reactive oxygen species. This suggests that Alternaria uses the active oxygen quencher mannitol to overcome host defence. On the other hand, pathogen attack was found to induce mannitol dehydrogenase-expression in tobacco, suggesting that plants counter this fungal defence system by catabolizing fungal mannitol (Jennings et al., 1998). Indeed, providing evidence for this hypothesis, it was demonstrated that the constitutive expression of a mannitol dehydrogenase gene in tobacco confers resistance against A. alternata, but not against non-mannitol secreting fungi (Jennings et al., 2002).

Using a transgenic approach, in a number of cases enhanced levels of resistance against Alternaria species were obtained. It was previously discussed that the expression of anti-apoptotic genes provides disease resistance, against Alternaria species amongst others, through blocking a programmed cell death response (Dickman et al., 2001; Lincoln et al., 2002). Constitutive expression of a radish defensin gene, a rubber tree chitin-binding lectin and a human lysozyme, conferred enhanced resistance against Alternaria spp. in tobacco, Indian mustard and carrot, respectively (Kanrar et al., 2002; Takaichi and Oeda, 2000; Terras et al., 1995). In addition, constitutive expression of an endochitinase gene isolated from the biocontrol fungus Trichoderma harzianum has resulted in enhanced Alternaria resistance in transgenic tobacco, potato and broccoli (Lorito et al., 1998; Mora and Earle, 2001). In addition, the treatment of plants with biocontrol organisms has in some cases resulted in enhanced resistance against Alternaria pathogens (Morita et al., 2003; Ton et al., 2002).

As should be evident from this pathogen profile, many aspects of the interaction between Alternaria species and their respective hosts are currently under investigation. The function and biosynthesis of host-specific toxins and melanins in particular has gained much attention, and considerable advances have been made. Finally, although the general strategies employed by the pathogenic strains to attack their hosts are similar, the responses of the various hosts against these attacks are very diverse.

ACKNOWLEDGEMENTS

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES

The author would like to thank Drs De Bolle, Thevissen and François and Ir. Van Hemelrijck for critical comments. Drs Barron, Bhatia, Timmer and Lopes are acknowledged for providing the Alternaria pictures. B.P.H.J.T. received a postdoctoral fellowship from the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’.

REFERENCES

  1. Top of page
  2. SUMMARY
  3. INTRODUCTION
  4. CLASSIFICATION
  5. MELANIN PRODUCTION
  6. THE INFECTION PROCESS
  7. TOXIN BIOSYNTHESIS
  8. MODE OF ACTION OF HOST-SPECIFIC TOXINS
  9. HOST DEFENCE AGAINST ALTERNARIA SPECIES
  10. ACKNOWLEDGEMENTS
  11. REFERENCES
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