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The wheat/Pyrenophora tritici-repentis interaction: progress towards an understanding of tan spot disease^†

^†This manuscript is an invited paper in recognition of the Outstanding Research Award granted to Dr. L. Lamari by the Canadian Phytopathological Society.

Abstract

Tan spot, caused by the fungus Pyrenophora tritici-repentis, is an important foliar disease of wheat. Eight races of P. tritici-repentis have been identified to date, based on their ability to cause necrosis and/or chlorosis on a set of wheat differential hosts. These symptoms result from the action of host-specific toxins, which are produced in different combinations by races of the fungus and mediate the interaction between host and pathogen. Ptr ToxA induces necrosis on sensitive wheat genotypes, and is a 13.2 kDa protein encoded by the ToxA gene. Ptr ToxB, encoded by the ToxB gene, is a 6.6 kDa, chlorosis-inducing protein. Ptr ToxC also induces chlorosis, but appears to be a non-proteinaceous, polar, non-ionic, low-molecular-mass molecule. In susceptible wheat, three dominant and independently inherited toxin-sensitivity genes have been identified, each controlling the reaction to a single toxin. Most studies have also found that sensitivity to the toxins and susceptibility to the producing races co-segregate, and appear to be controlled by the same genes. Therefore, the tan spot pathosystem conforms to the toxin model of host–pathogen interactions, in which the compatible interaction is the basis for specificity (as opposed to the incompatible interaction in the classical gene-for-gene model). It is likely that P. tritici-repentis produces additional toxins or pathogenicity factors, but these remain to be identified.

Résumé: La tache helminthosporienne, causée par le champignon Pyrenophora tritici-repentis, est une grave maladie foliaire du blé. Huit races de P. tritici-repentis ont été identifiées à ce jour, en se basant sur leur capacité à provoquer la nécrose ou la chlorose dans un groupe d'hôtes différentiels chez le blé. Ces symptômes apparaissent à la suite de l'effet de toxines spécifiques de l'hôte qui sont produites selon différentes combinaisons par les races de champignons et qui médient les interactions entre l'hôte et l'agent pathogène. Ptr ToxA est une protéine 13.2 kDa encodée par le gène ToxA, qui provoque la nécrose chez les génotypes de blé sensibles. Ptr ToxB, encodé par le gène ToxB, est une protéine 6.6 kDa qui provoque la chlorose. Ptr ToxC provoque également la chlorose, mais il semble s'agir d'une molécule non protéique, polaire, non ionique et de faible poids moléculaire. Chez les variétés de blé réceptives, trois gènes dominants et transmis de façon indépendante, responsables de la sensibilité à la toxine, ont été identifiés, chacun régissant la réaction à une seule toxine. La plupart des études ont également trouvé que la sensibilité aux toxines et la réceptivité aux races qui les produisent affichent une coségrégation et semblent être régies par les mêmes gènes. Par conséquent, le pathosystème de la tache helminthosporienne est conforme au modèle de la toxine relatif aux interactions hôte-pathogène, selon lequel l'interaction compatible est le fondement de la spécificité (par opposition à l'interaction incompatible du modèle classique gène pour gène). Il est probable que P. tritici-repentis produise des toxines de plus ou des facteurs de pathogénicité, mais il nous reste à les identifier.

Keywords:

• effectors
• host-pathogen interactions
• tan spot
• toxins
• Pyrenophora tritici-repentis
• wheat

Mots clés:

• blé
• effecteurs
• interactions hôte-pathogène
• tache helminthosporienne
• toxines
• Pyrenophora tritici-repentis

Introduction

Tan spot, caused by the necrotrophic fungus Pyrenophora tritici-repentis (Died.) Drechs. (anamorph: Drechslera tritici-repentis (Died.) Shoem.), is a major foliar disease of wheat worldwide, causing yield losses that range from 3 to 50% (da Luz & Hosford, 1980; Shabber & Bockus, 1988). On susceptible host genotypes, P. tritici-repentis causes the development of tan-coloured necrotic lesions, which are often surrounded by chlorotic borders or haloes that give the disease its other name, yellow spot. The fungus survives on infected stubble on the soil surface, and as a consequence, the widespread adoption of conservation tillage practices that retain crop residues has led to an increased incidence of tan spot in recent decades. This increased prevalence of the disease has also resulted in greater interest in the P. tritici-repentis/wheat interaction, with researchers working to elucidate the mechanisms of tan spot development, pathogen virulence and host susceptibility. As a result of these efforts, significant strides have been made towards improving understanding of tan spot disease and the nature of this pathosystem. The current review is intended to highlight some of these advances and their implications.

The race structure of Pyrenophora tritici-repentis

Early studies described the variation in virulence of P. tritici-repentis in strictly quantitative terms. Parameters such as lesion size (Misra & Singh, 1972), per cent leaf necrosis (Schilder & Bergstrom, 1990), and lesion number and per cent infection (da Luz & Hosford, 1980) were used to discern variation in isolates of the fungus. However, these quantitative approaches made limited contributions to knowledge of host–parasite relations in the tan spot pathosystem. It was not until Lamari & Bernier (1989a, 1991) demonstrated that the necrosis and chlorosis symptoms associated with tan spot are in fact distinct, and result from highly specific interactions between host and pathogen, that the underlying basis of virulence and symptom development began to be understood. This allowed for the initial classification of P. tritici-repentis isolates into pathotypes, according to their capacity to induce necrosis and/or chlorosis on the leaves of selected wheat genotypes (Lamari & Bernier 1989a). However, this symptom-based classification system could accommodate a maximum of only four groups: pathotype 1 (necrosis⁺chlorosis⁺), pathotype 2 (nec⁺chl⁻), pathotype 3 (nec⁻chl⁺), and pathotype 4 (nec ⁻ chl ⁻ ).

The limitations of the pathotype classification scheme became evident with the identification of isolates of P. tritici-repentis that could induce the same symptom, but on different host genotypes. To accommodate the new virulence patterns, a race-based classification was developed, in which isolates were classified according to their virulence pattern on a wheat differential set (Lamari et al., 1995). This system was adopted by most research groups and is widely used today. Eight races of P. tritici-repentis are currently recognized, which have been numbered chronologically according to the order in which they were first reported (Table 1). Thus, races 1 to 4 correspond to the original pathotypes 1 to 4 (Lamari et al., 1995). These races were originally identified from North American collections of the pathogen, where races 1 and 2 are predominant (Lamari et al., 1998), but have subsequently also been found in the wheat centre of diversity (Lamari et al., 2005). Race 5 was first reported from Algeria (Lamari et al., 1995), and later from the United States (Ali et al., 1999), Canada (Strelkov et al., 2002), Syria and Azerbaijan (Lamari et al., 2005). To date, race 6 has been found only in North Africa (Strelkov et al., 2002), whilst races 7 and 8 of P. tritici-repentis have been reported only from the Middle East and the Caucasus (Strelkov et al., 2002; Lamari et al., 2003, 2005). The greatest diversity in terms of pathogen race composition occurs in these latter regions, which constitute the wheat centre of diversity; this is expected, since the centre of origin of a host may also represent the centre of greatest variability for its pathogens (Vavilov, 1951). The global distribution of tan spot, along with the races identified in different regions, is illustrated in Fig. 1. In addition to these eight well-characterized races, there have been preliminary reports of other races (Manning et al., 2002; Meinhardt et al., 2003), but these have not been fully described and await publication in peer-reviewed journals.

Table 1.  Reactions of six wheat differential hosts to the eight characterized races of Pyrenophora tritici-repentis

Host genotype^1	Race 1	Race 2	Race 3	Race 4	Race 5	Race 6	Race 7	Race 8
‘Glenlea’	S (N)^2	S (N)	R	R	R	R	S (N)	S (N)
6B662	R	R	R	R	S (C)	S (C)	S (C)	S (C)
6B365	S (C)	R	S (C)	R	R	S (C)	R	S (C)
‘Salamouni’	R	R	R	R	R	R	R	R
‘Coulter’	S (N)	S (N)	S (N)	R	S (N)	S (N)	S (N)	S (N)
4B1149	R	R	R	R	R	R	R	R
Notes: ^1All host genotypes listed are hexaploid except for ‘Coulter’ and line 4B1149, which are tetraploid.
^2S (N) = susceptible (necrotic reaction); S (C) =  susceptible (chlorotic reaction); R = resistant.

Fig. 1. Global distribution of tan spot [Pyrenophora tritici-repentis] of wheat, with countries in which the disease has been reported highlighted in grey. The numbers on the map correspond to races identified in the different regions. In North America, races 1 and 2 represent more than 90% of the total population of the pathogen.

Limitations of the current differentials

The race classification of P. tritici-repentis is based on the reaction of the six wheat lines/cultivars that constitute the differential set (Table 1). However, of these, only three genotypes (‘Glenlea’ and lines 6B365 and 6B662) are effective for differentiating the eight recognized races of P. tritici-repentis (Lamari et al., 2003). Given the limited composition of this differential set, Lamari et al. (2005) noted that it may not adequately reflect the full diversity of pathogen populations, especially in important regions such as the wheat centre of origin. They suggested that, in order to fully evaluate diversity in P. tritici-repentis from Central Asia, the Caucasus, North Africa and/or the Fertile Crescent, the inclusion of wheat landraces or varieties from these regions should be considered (Lamari et al., 2005). Landraces or varieties with unknown pedigrees, however, may be genetically heterogeneous, which could result in variable disease reactions, leading to confusion and inconsistent race classifications. Thus, caution should be used in selecting wheat genotypes for inclusion as new differentials, with the consistency of their reaction to inoculation validated with available isolates of P. tritici-repentis.

An alternative approach to fully capture the extent of physiological variation in the pathogen was proposed by Andrie et al. (2007), who suggested a combination of phenotypic and genotypic characterization for the identification of races of P. tritici-repentis. Using multiplex PCR, these authors demonstrated that some fungal isolates producing disease phenotypes similar to those of known races were genotypically distinct from those races. However, this method was based on the PCR amplification of genes encoding two host-specific toxins (Ptr ToxA and Ptr ToxB; Andrie et al., 2007), homologues of which also exist in other species and/or races (Martinez et al., 2004; Friesen et al., 2006; Strelkov et al., 2006; Andrie et al., 2008). Thus, it would be difficult to rule out amplification of other forms of these genes when evaluating uncharacterized isolates of the fungus. As a way of addressing this concern, Andrie et al. (2007) recommended that all positive multiplex PCR results be confirmed by toxin isolation from culture filtrates or, alternatively, via Western blotting analysis. However, the need to conduct a multiplex PCR followed by toxin purification or Western blotting, in addition to the inoculation of isolates on a host differential set, likely precludes routine application of this procedure in most labs. The fact that the gene(s) involved in the synthesis of a third host-specific toxin (Ptr ToxC) are unknown also represents a significant gap in the utility of this protocol. Nevertheless, such an approach could be useful to confirm race designations. Indeed, the first reports describing races 6, 7 and 8 of P. tritici-repentis included not only data on the reaction of the host differentials, but also on toxin production and/or the occurrence of the ToxA and ToxB genes (Strelkov et al., 2002; Lamari et al., 2003).

The Ptr toxins and virulence of the pathogen

The virulence of the races of P. tritici-repentis is defined based on the differential production of at least three host-specific toxins, which were alluded to in the previous section. These toxins, termed Ptr ToxA, Ptr ToxB and Ptr ToxC (Ciuffetti et al., 1998), are largely responsible for the necrosis and chlorosis associated with tan spot and serve as pathogenicity (Strelkov & Lamari, 2003) or virulence factors (Friesen et al., 2003) for the fungus (Table 2). Races 2, 3 and 5 produce only one toxin each (Ptr ToxA, Ptr ToxC and Ptr ToxB, respectively) and are therefore considered the ‘basic’ races, while races 1, 6, 7 and 8 each produce more than one toxin and are considered ‘composite’ races; race 1 produces Ptr ToxA and Ptr ToxC, race 6 produces Ptr ToxB and Ptr ToxC, race 7 produces Ptr ToxA and Ptr ToxB, and race 8 produces all three toxins (Strelkov et al., 2002; Lamari et al., 2003). In contrast, race 4 does not produce any active toxins and is therefore avirulent. Interestingly, the levels of numerous other proteins implicated in microbial virulence are also significantly lower in race 4, suggesting a reduced general pathogenic ability in this race irrespective of toxin production (Cao et al., 2009).

Table 2.  Production of host-specific toxins by the eight characterized races of Pyrenophora tritici-repentis

Race	Toxins	Toxin genes or homologues
1	Ptr ToxA, Ptr ToxC	ToxA, (ToxC?)^1
2	Ptr ToxA	ToxA
3	Ptr ToxC	ToxB homologue^2, (ToxC?)
4	none	ToxB homologue^3 (= toxb)
5	Ptr ToxB	ToxB
6	Ptr ToxB, Ptr ToxC	ToxB, (ToxC?)
7	Ptr ToxA, Ptr ToxB	ToxA, ToxB
8	Ptr ToxA, Ptr ToxB, Ptr ToxC	ToxA, ToxB, (ToxC?)
Notes: ^1The gene(s) involved in the synthesis of Ptr ToxC are unknown.
^2Although race 3 possesses a ToxB homologue, it does not exhibit any Ptr ToxB activity (Strelkov et al., 2006).
^3Although race 4 possesses a ToxB homologue, it does not exhibit any Ptr ToxB activity (Martinez et al., 2004; Strelkov et al., 2006); the race 4 homologue was termed toxb by Martinez et al. (2004).

Ptr ToxA and the development of host-specific necrosis

Ptr ToxA was the first of the toxins to be isolated, and induces necrosis on sensitive wheat genotypes (Ballance et al., 1989; Tomás et al., 1990; Tuori et al., 1995; Zhang et al., 1997). It is a small, secreted protein, encoded by the ToxA gene, which is of 13.2 kDa mass when mature (Ballance et al., 1996; Ciuffetti et al., 1997). The exact mechanism of Ptr ToxA action has not been elucidated. However, Ptr ToxA-induced necrosis requires light (Manning & Ciuffetti, 2005) and an active host metabolism (Kwon et al., 1998). Moreover, the toxin is internalized only in cells of toxin-sensitive wheat cultivars, where it localizes to cytoplasmic compartments and chloroplasts, suggesting that sensitivity to Ptr ToxA is related to protein import (Manning & Ciuffetti, 2005).

The ToxA gene is highly conserved in isolates of P. tritici-repentis from geographically diverse regions (Ballance et al., 1996; Ciuffetti et al., 1997; Friesen et al., 2006). However, homologues of ToxA have also recently been identified in Stagonospora nodorum (Berk.) E. Castell. & Germano, the causal agent of leaf and glume blotch of wheat (Friesen et al., 2006). In contrast to the conserved nature of the gene in P. tritici-repentis, the homologues in S. nodorum exhibit a high level of nucleotide diversity, which, along with evidence of inter-specific gene transfer, led Friesen et al. (2006) to suggest that acquisition of the ToxA gene by P. tritici-repentis resulted in the emergence of tan spot as a major disease problem. Given that the necrosis associated with tan spot was not described until the early 1940s, Friesen et al. (2006) further hypothesized that this inter-specific gene transfer transpired just prior to 1941. However, if transfer of this gene did occur less than 70 years ago, it is difficult to envision how isolates of P. tritici-repentis carrying ToxA would have become so widely disseminated so quickly, particularly in the highly diverse pathogen populations from the host centre of diversity (Lamari et al., 2003, 2005). Thus, while it is very likely that inter-specific transfer of ToxA from S. nodorum to P. tritici-repentis did occur, this event may have taken place at a much earlier time (Strelkov & Lamari, 2003).

Ptr ToxB and the development of host-specific chlorosis

In addition to Ptr ToxA, P. tritici-repentis also produces Ptr ToxB, another proteinaceous, host-specific toxin, the mature form of which is 6.6 kDa in mass (Strelkov et al., 1999). Ptr ToxB induces chlorosis on sensitive wheat genotypes through the light-dependent degradation of chlorophyll, likely as a consequence of a direct or indirect inhibition of photosynthetic processes (Strelkov et al., 1998). However, the exact mode of Ptr ToxB action remains to be elucidated. The toxin is encoded by ToxB, which unlike ToxA, is a multicopy gene that is variable in sequence amongst races of P. tritici-repentis (Martinez et al., 2004; Strelkov et al., 2006). Homologues of ToxB have even been identified in isolates representing races 3 and 4, which possess no detectable Ptr ToxB activity (Martinez et al., 2004; Strelkov et al., 2006). The role of these homologues, however, remains unclear. Although the form of ToxB in avirulent race 4 is expressed in low quantities (Amaike et al., 2008), the protein it encodes induces only trace levels of chlorosis on toxin-sensitive wheat leaves (Kim & Strelkov, 2007). Andrie et al. (2008) suggested that the ToxB homologue in race 4 (which they termed toxb) may be evolving towards a fate as a pseudogene, although it is not clear why, if this is the case, race 4 isolates from geographically diverse regions maintain identical forms of the gene (Strelkov et al., 2006). In addition, roles for ToxB homologues in the infection of other hosts (Martinez et al., 2004), basic pathogenic fitness (Amaike et al., 2008), or even in primary metabolism (Andrie et al., 2008) have been suggested. Indeed, the recent identification of ToxB homologues in Pyrenophora bromi (Died.) Drechsler and other members of the Pleosporaceae (Andrie et al., 2008) supports a wider role for this gene; it also suggests that ToxB may have arisen in an early ancestor of the Ascomycota (Andrie et al., 2008), and is consistent with the hypothesis that P. tritici-repentis and its many toxins evolved on grass species before moving to its wheat host (Strelkov & Lamari, 2003).

Ptr ToxC, another chlorosis-inducing toxin from Pyrenophora tritici-repentis

The third host-specific toxin produced by P. tritici-repentis has been termed Ptr ToxC. Like Ptr ToxB, Ptr ToxC also induces chlorosis, but on different wheat genotypes. While Ptr ToxC has been predicted by a number of studies (Lamari & Bernier, 1991; Gamba & Lamari, 1998; Gamba et al., 1998), it has not been purified to homogeneity nor has it been fully characterized. Nevertheless, based on analysis of a partially purified toxic principle believed to be Ptr ToxC, the toxin appears to be a non-ionic, polar, low-molecular-mass molecule (Effertz et al., 2002), and is therefore quite distinct from the proteinaceous Ptr ToxA and Ptr ToxB. Although knowledge regarding the exact nature of Ptr ToxC is lacking, it is clear from genetic studies that this toxin also functions as an important pathogenicity factor for P. tritici-repentis (Lamari & Bernier, 1991; Gamba & Lamari, 1998; Gamba et al., 1998).

Other toxins?

The host-specific toxins identified to date do not, on their own, account for all of the virulence in P. tritici-repentis (Strelkov & Lamari, 2003). In a study of the inheritance of resistance to tan spot in selected durum wheat genotypes, Gamba & Lamari (1998) suggested that, in addition to Ptr ToxA and Ptr ToxC, race 1 isolates of the fungus produce another factor that selectively induces chlorosis on a durum wheat line. The same authors suggested that isolates of races 3 and 5 produce toxic constituents that cause a highly specific necrosis on this line (Gamba & Lamari, 1998). Although there is strong genetic evidence for the production of these factors by P. tritici-repentis, their existence has not been demonstrated. There have been preliminary reports of other putative host-selective toxins from P. tritici-repentis. Meinhardt et al. (2003) identified a ‘Ptr ToxD’ that induced chlorosis on certain wheat genotypes, while Ciuffetti et al. (2003) reported another necrosis-inducing toxin, distinct from Ptr ToxA, which they also termed ‘Ptr ToxD’. These toxins, however, await full characterization and description in the refereed literature. The verification of proteins, metabolites or other compounds as host-specific toxins or pathogenicity factors requires not only that these molecules be shown to induce symptoms in a genotype-specific manner, but also that host sensitivity to these compounds and susceptibility to the producing isolates co-segregate in the host.

In addition to host-specific toxins, P. tritici-repentis produces a number of non-specific phytotoxic compounds, including the necrosis-inducing spirocyclic lactams (Hallock et al., 1993) and the anthraquinone catenarin (Bouras & Strelkov, 2008). The role of these compounds in symptom development appears to be small, relative to the host-specific toxins, although they might contribute to the aggressiveness of virulent isolates (Bouras & Strelkov, 2008). Nevertheless, their production by P. tritici-repentis could mask specific interactions between host and pathogen, serving to obscure results.

Host reaction and the one-to-one relationship in tan spot of wheat

Genetic studies on wheat have confirmed that the tan spot pathosystem conforms to the toxin model of host-pathogen relations, in which the compatible interaction is the basis for specificity (as opposed to the incompatible interaction in the classical gene-for-gene model) (Loegering, 1978). Three dominant and independently inherited toxin-sensitivity genes have been identified in susceptible wheat, each controlling the reaction to a single toxin (Gamba et al., 1998; Effertz et al., 2002; Friesen & Faris, 2004). Most studies have also found that sensitivity to the toxins and susceptibility to the producing races co-segregate, and appear to be controlled by the same genes (Lamari & Bernier, 1991; Gamba & Lamari, 1998; Gamba et al., 1998; Singh & Hughes, 2006). In addition, the acquisition of Ptr ToxA-producing ability was shown to be a sufficient condition for virulence in P. tritici-repentis (Ciuffetti et al., 1997). However, one study found that reaction to Ptr ToxA accounted for only 24% of the resistance to race 2 (Friesen et al., 2003) – a discrepancy that could reflect the differential production of non-specific toxins or secondary metabolites by isolates of the fungus (Hallock et al., 1993; Bouras & Strelkov, 2008), and/or the presence of uncharacterized susceptibility factors in the host (Strelkov & Lamari, 2003). Nonetheless, the preponderance of the evidence strongly supports a model in which the products encoded by the toxin-sensitivity genes in the host interact with the fungal toxins in a highly specific manner, resulting in a compatible interaction and the development of disease. In this context, the eight races of P. tritici-repentis identified to date account for all of the virulence patterns expected from three toxins matching three loci in the host (Lamari et al., 2003).

Conclusions

The increased prevalence of tan spot has spawned a great deal of interest in the wheat/P. tritici-repentis interaction, which has in turn led to significant advances in understanding of this disease. Among the most important of these advances was the realization that the necrosis and chlorosis symptoms associated with tan spot result from highly specific interactions between the host and pathogen; this enabled the rational classification of fungal isolates into races, as well as the identification of the host-specific toxins or effectors that mediate this interaction. The tan spot pathosystem became the first to be recognized in which a single pathogen produces multiple host-specific toxins that selectively attack different genotypes of a single host species, in a mirror image of the classical gene-for-gene relationship. These findings have led not only to an increased understanding of tan spot of wheat, but have also contributed to knowledge of host–pathogen relationships in general. Indeed, homologues of ToxA and ToxB have now been found in other fungal pathogens, while the Stagonospora nodorum/wheat interaction appears to follow a model very similar to that of tan spot (Friesen et al., 2007). Despite these great strides, additional research is needed, including work on the identification of new races and toxins, the elucidation of the mechanisms of toxin action, and the molecular basis of host susceptibility/resistance. These efforts will be aided by the recent sequencing of the P. tritici-repentis genome (www.broadinstitute.org), and by the progress that has already been made on this important pathosystem.

Notes

^†This manuscript is an invited paper in recognition of the Outstanding Research Award granted to Dr. L. Lamari by the Canadian Phytopathological Society.