SnTox5–Snn5: a novel Stagonospora nodorum effector–wheat gene interaction and its relationship with the SnToxA–Tsn1 and SnTox3–Snn3–B1 interactions

The Stagonospora nodorum–wheat interaction involves multiple pathogen‐produced necrotrophic effectors that interact directly or indirectly with specific host gene products to induce the disease Stagonospora nodorum blotch (SNB). Here, we used a tetraploid wheat mapping population to identify and characterize a sixth effector–host gene interaction in the wheat–S. nodorum system. Initial characterization of the effector SnTox5 indicated that it is a proteinaceous necrotrophic effector that induces necrosis on host lines harbouring the Snn5 sensitivity gene, which was mapped to the long arm of wheat chromosome 4B. On the basis of ultrafiltration, SnTox5 is probably in the size range 10–30 kDa. Analysis of SNB development in the mapping population indicated that the SnTox5–Snn5 interaction explains 37%–63% of the variation, demonstrating that this interaction plays a significant role in disease development. When the SnTox5–Snn5 and SnToxA–Tsn1 interactions occurred together, the level of SNB was increased significantly. Similar to several other interactions in this system, the SnTox5–Snn5 interaction is light dependent, suggesting that multiple interactions may exploit the same pathways to cause disease.     

Identification and characterization of a novel host–toxin interaction in the wheat–<Emphasis Type="Italic">Stagonospora nodorum</Emphasis> pathosystem

Stagonospora nodorum, casual agent of Stagonospora nodorum blotch (SNB) of wheat, produces a number of host-selective toxins (HSTs) known to be important in disease. To date, four HSTs and corresponding host sensitivity genes have been reported, and all four host–toxin interactions are significant factors in the development of disease. Here, we describe the identification and partial characterization of a fifth S. nodorum produced HST designated SnTox4. The toxin, estimated to be 10–30 kDa in size, was found to be proteinaceous in nature. Sensitivity to SnTox4 is governed by a single dominant gene, designated Snn4, which mapped to the short arm of wheat chromosome 1A in a recombinant inbred (RI) population. The compatible Snn4–SnTox4 interaction is light dependent and results in a mottled necrotic reaction, which is different from the severe necrosis that results from other host–toxin interactions in the wheat–S. nodorum pathosystem. QTL analysis in a population of 200 RI lines derived from the Swiss winter wheat varieties Arina and Forno revealed a major QTL for SNB susceptibility that coincided with the Snn4 locus. This QTL, designated QSnb.fcu-1A, explained 41.0% of the variation in disease on leaves of seedlings indicating that a compatible Snn4–SnTox4 interaction plays a major role in the development of SNB in this population. Additional minor QTL detected on the short arms of chromosomes 2A and 3A accounted for 5.4 and 6.0% of the variation, respectively. The effects of the three QTL were largely additive, and together they explained 50% of the total phenotypic variation. These results provide further evidence that host–toxin interactions in the wheat–S. nodorum pathosystem follow an inverse gene-for-gene model.     

Genetic analysis of disease susceptibility contributed by the compatible Tsn1–SnToxA and Snn1–SnTox1 interactions in the wheat-Stagonospora nodorum pathosystem

Stagonospora nodorum is a foliar pathogen of wheat that produces several host-selective toxins (HSTs) and causes the disease Stagonospora nodorum blotch (SNB). The wheat genes Snn1 and Tsn1 confer sensitivity to the HSTs SnTox1 and SnToxA, respectively. The objectives of this study were to dissect, quantify, and compare the effects of compatible Snn1–SnTox1 and Tsn1–SnToxA interactions on susceptibility in the wheat-S. nodorum pathosystem. Inoculation of a wheat doubled haploid population that segregates for both Snn1 and Tsn1 with an S. nodorum isolate that produces both SnTox1 and SnToxA indicated that both interactions were strongly associated with SNB susceptibility. The Snn1–SnTox1 and Tsn1–SnToxA interactions explained 22 and 28% of the variation in disease, respectively, and together they explained 48% indicating that their effects are largely additive. The Snn1–SnTox1 interaction accounted for 50% of the variation when the population was inoculated with an S. nodorum strain where the SnToxA gene had been mutated, eliminating the Tsn1–SnToxA interaction. These results support the theory that the wheat-S. nodorum pathosystem is largely based on multiple host–toxin interactions that follow an inverse gene-for-gene scenario at the host–toxin interface, but disease exhibits quantitative variation due to the mainly additive nature of compatible interactions. The elimination of either Snn1 or Tsn1 toxin sensitivity alleles resulted in decreased susceptibility, but the elimination of both interactions was required to obtain high levels of resistance. We propose the use of molecular markers to select against Snn1, Tsn1, and other toxin sensitivity alleles to develop wheat varieties with high levels of SNB resistance.     

Application of PCR based diagnostics in the exploration of Parastagonospora nodorum prevalence in wheat growing regions of Himachal Pradesh

Stagonospora leaf and glume blotch (SLGB) of wheat caused by Parastagonospora nodorum (formerly Stagonospora nodorum) has recently emerged as a major problem in changing climatic conditions of Himachal Pradesh (HP), especially during delayed winter rains. In the present studies symptomatology, morpho-cultural as well as molecular marker based identification showed the prevalence of disease in the state and conclusively proved that leaf and glume blotch of wheat is caused by P. nodorum. The test pathogen showed 100% homology with other reported P. nodorum isolates by rDNA (ribosomal DNA) analysis. In addition, the amplification of rDNA region of 36 P. nodorum isolates representing various agro-ecological areas of HP and one infected wheat leaf sample generated an amplicon of ~ 449-bp with JB433 (5′-ACACTCAGTAGTTTACTACT-3′) and JB434 (5′-TGTGCTGCGCTTCAATA-3′) P. nodorum specific primer pair whereas no amplification was observed with the genomic DNA of Septoria titici, Stemphylium vesicarium and healthy wheat leaf sample. This study on integration of morpho-cultural and microscopic methods along with PCR based technique could form basis for routine diagnosis of the SLGB in wheat samples during early growth stages of crop in the seed production fields.

     

Host-selective toxins produced by <Emphasis Type="Italic">Stagonospora nodorum</Emphasis> confer disease susceptibility in adult wheat plants under field conditions

Stagonospora nodorum, causal agent of Stagonospora nodorum blotch (SNB), is a destructive pathogen of wheat worldwide. As is true for many necrotrophic host–pathogen systems, the wheat-S. nodorum system is complex and resistance to SNB is usually quantitatively inherited. We recently showed that S. nodorum produces at least four proteinaceous host-selective toxins that interact with dominant host sensitivity/susceptibility gene products to induce SNB in seedlings. Here, we evaluated a population of wheat recombinant inbred lines that segregates for Tsn1, Snn2, and Snn3, which confer sensitivity to the toxins SnToxA, SnTox2, and SnTox3, respectively, to determine if compatible host–toxin interactions are associated with adult plant susceptibility to SNB foliar disease under field conditions. Artificial inoculation of the population in 2 years and two locations with a fungal isolate known to produce SnToxA and SnTox2 indicated that compatible SnToxA–Tsn1 and SnTox2–Snn2 interactions accounted for as much as 18 and 15% of the variation in disease severity on the flag leaf, respectively. As previously reported for seedlings, the effects of these two interactions in conferring adult plant susceptibility were largely additive. Additional adult plant resistance QTLs were identified on chromosomes 1B, 4B, and 5A, of which, the 1B and 5A QTLs were previously reported to be associated with seedling resistance to SNB. Therefore, in this population, some of the same QTLs are responsible for seedling and adult plant resistance/susceptibility. This is the first report showing that host-selective toxins confer susceptibility of adult plants to SNB, further substantiating the importance of compatible toxin–host interactions in the wheat-S. nodorum pathosystem. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Diagnostics of phytopathogen fungi <Emphasis Type="Italic">Septoria tritici</Emphasis> and <Emphasis Type="Italic">Stagonospora nodorum</Emphasis> by fluorescent amplification-based specific hybridization (FLASH) PCR

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola) and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.     

Disease spread of non-specialised fungal pathogens from inoculated point sources in intraspecific mixed stands of cereal cultivars

Spread of Septoria nodorum from inoculated point sources was examined in pure stands and mixtures of two spring wheat cultivars Kolibri and Maris Butler. Gradients in disease were observed soon after inoculation; the presence of the more resistant cultivar (Maris Butler) in the mixtures retarded the outward spread of disease compared with the susceptible pure stand (Kolibri). Regression analysis suggested that gradients in incidence were influenced by nearness to the source whereas gradients in severity were not. Spread of disease was also examined in pure stands and mixtures for the host/pathogen combinations, winter wheat (cvs Maris Huntsman and Maris Ranger)/S. nodorum and winter barley (cvs Maris Otter and Hoppel)/Rhynchosporium secalis. In contrast to the spring wheat experiment, no gradients were observed; explanations for the uniform distribution of disease were (a) extensive spread prior to the period of assessment, (b) no physical barrier to dispersal due to the juvenile growth stage of the crop and (c) exhaustion of the point-source.     

Identification of molecular markers for resistance to Septoria nodorum blotch in durum wheat

The development of Septoria nodorum blotch-resistant cultivars has become a high priority objective for durum wheat breeding programs. Marker-assisted selection enables breeders to improve selection efficiency. In order to develop markers for resistance to Septoria nodorum blotch, a set of F₅ recombinant inbred lines, derived from the crosses Sceptre/3–6, Sceptre/S9–10 and Sceptre/S12–1, was developed based on the F₂-derived family method. Two RAPD markers, designated UBC521₆₅₀ and RC37₅₁₀, were detected by bulked segregant analysis and located approximately 15 and 13.1 centiMorgans (cM) from the resistance gene snbTM, respectively. A SCAR marker was also successfully developed for marker-assisted selection in breeding programs based on the sequence of the RAPD marker UBC521₆₅₀. This is the first report of DNA-based markers linked to resistance for Septoria nodorum blotch in durum wheat. Received: 8 March 2000 / Accepted: 25 June 2000     

Comparative studies on the activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in the leaves of triticale and wheat infected with Stagonospora nodorum

The activities of chitinase, β-1,3-glucanase, peroxidase and phenylalanine ammonia lyase, constitutive and induced by Stagonospora nodorum were examined in the 10 – 14 day old seedlings of three triticale and two wheat cultivars under controlled environmental conditions and in flag leaves of two triticale cultivars in the field. Two S. nodorum isolates of different virulence were used. Both the constitutive and induced activities in triticale and wheat depended on genotype and in triticale the effect of growth conditions was also evidenced. The constitutive activities of chitinase, β-1,3-glucanase and peroxidase were several fold lower in flag triticale leaves in plants from the field than in the seedlings, growing under controlled conditions, but induction in the infected flag leaves was significantly more pronounced. In triticale genotypic differences in the response to infection were revealed only upon inoculation by S. nodorum isolate of higher virulence. The enzymatic activities increased several fold during successive days after the infection except for phenylalanine ammonia lyase. Induction of this enzyme was only transient and the activity decreased 48 or 96 h after infection when the activities of other enzymes were rising. In flag leaves in the field this activity was differentiated only after infection with more a virulent strain. A tendency appeared in triticale seedlings for association of the resistance to the pathogen with lower enzymatic constitutive activities. This relationship became more evident in triticale infected by S. nodorum and may imply that although the investigated enzymes are certainly involved in general, non-specific defense mechanism, they do not decide on the resistance to pathogen at least in the early stages of infection and cooperate with other factors in the complex pathogen-plant interaction. One can also assume that the enzymatic activities are associated with severity of infection rather than resistance to pathogen.     

Molecular cytogenetic characterization of four partial wheat-Thinopyrum ponticum amphiploids and their reactions to Fusarium head blight, tan spot, and Stagonospora nodorum blotch

Four wheat (Triticum aestivum L.)-Thinopyrum ponticum derivatives SS5 (PI604926), SS156 (PI604947), SS363 (PI604970), and SS660 (PI604879), were identified as resistant to Fusarium head blight (FHB), a serious fungal disease of wheat worldwide. Seedling reactions to tan spot and Stagonospora nodorum blotch (SNB), two important foliar diseases of wheat, suggest that these four derivatives are resistant to tan spot and two of them (SS5 and SS156) are resistant to SNB. Fluorescent genomic in situ hybridization (FGISH) patterns of mitotic chromosomes indicate that these four derivatives are partial wheat-Th. ponticum amphiploids, each with a total of 56 chromosomes, though with different amounts of Th. ponticum chromatin. These four amphiploids were hybridized with each other to determine homology between the Th. ponticum genomes in each of the amphiploids. Analysis of chromosome pairing in the F₁ hybrids using FGISH suggests that each amphiploid carries a similar set of Th. ponticum chromosomes. These wheat-Th. ponticum amphiploids represent a potential novel source of resistance to FHB, tan spot, and SNB for wheat breeding.     

Isolation and characterization of EST‐derived microsatellite loci from the fungal wheat pathogen Phaeosphaeria nodorum

Eleven polymorphic microsatellite loci and one minisatellite locus originating from expressed sequence tag (EST) libraries of Phaeosphaeria (syn. Stagonospora) nodorum were isolated and characterized. The satellite markers were used to genotype isolates from field populations collected in China, North America and South Africa. The number of alleles per locus ranged from two to 15. Genotype diversity ranged from 87.5 to 95.3 and gene diversity from 0.1 to 0.8. The variable levels of polymorphism within and among populations of P. nodorum renders these 12 satellite loci ideal markers for population genetic analysis of P. nodorum.     

Effects of alien cytoplasm substitution on the response of wheat cultivars to Septoria nodorum

The effects of alien cytoplasm substitution on the response of wheat to Septoria nodorum were studied, using alloplasmic series of two cultivars, Chris and Selkirk. In general, cytoplasmic substitution caused unidirectional effects on Septoria-response, alloplasmic lines of both cultivars expressing lower levels of partial resistance (in leaf and head tissue) but higher levels of yield tolerance than the corresponding euplasmic line. The reduced resistance in alloplasmics was closely associated with reduced incubation periods of Septoria infection in both leaf and head tissue. Cytoplasmic substitution resulted in increased yield tolerance to Septoria-infection in both the non-tolerant Selkirk and the relatively tolerant Chris. Unlike their effects on partial resistance, specific cytoplasms exerted similar effects on tolerance in the two parental cultivars, several cytoplasms of the D plasmatype being particularly effective in increasing Septoria-tolevance. The potential for the development of Septoria-toterant cultivars by the incorporation of alien cytoplasms is discussed, in view of the observed neutral effects of D plasmatype cytoplasms on other agronomic traits.     

Effect of Ploidy and Flowering Type of Red Clover Cultivars and of Isolate Origin on Severity of Clover Rot, Sclerotinia trifoliorum

Two complementary experiments were conducted in a controlled environment to elucidate the interactions between the fungus Sclerotinia trifoliorum Erikss. and red clover (Trifolium pratense L.). In one of these studies, two hardened diploid red clover cultivars (cvs) were inoculated with 20 isolates of S. trifoliorum of various geographic origins. In the other study, 20 red clover cvs, diploid or tetraploid, including late and medium‐late flowering types, were inoculated with two isolates of the fungus. Prior to inoculation, some plants were hardened by subjecting them to a low temperature and light treatment mimicking autumn conditions. Late flowering cvs were found more resistant than medium‐late ones. Isolates collected in the northern region, where late cvs are grown, were significantly more aggressive than isolates from southern locations, where medium‐late cvs are more prevalent. Such an adaptation has not previously been reported for this fungus. This is the first report concerning flowering type and resistance in red clover. Tetraploids were generally not more resistant than diploids. A hardening procedure for red clover plants was found to be a prerequisite for detecting the differences in disease resistance.     

Virulence of Septoria nodorum as Affected by Passage Through Detached Leaves of Wheat and Barley Cultivars1

Abstract Three genetically marked, single–spore isolates of Septoria nodorum from wheat were passed through detached leaves of wheat cvs Blueboy and Coker 747 and the barley cv. Boone to produce three sub–isolates per original isolate. Each sub–isolate was cultured for three pycnidiospore generations on its respective host. Virulence of each sub–isolate on detached leaves of Blueboy, Caldwell, Coker 747, and NK81W701 wheat, and Boone and Surry barley was compared with that of the original single–spore isolate from which it was derived. In most cases, sub–isolates passed through wheat were significantly more virulent than the originals on wheat cultivars. They also were more virulent to barley than the original isolates but they were less virulent to barley than to wheat cultivars. Isolate × cultivar interactions were statistically significant (P < .0001) for isolates passed through wheat or barley and were greater than isolate × cultivar interactions among the original isolates. In seven of eight isolates passed through wheat or barley, only the original genetic marker was recovered after three generations, indicating that cross–contamination could not account for the observed change of virulence. In the single case of apparent contamination, of a sub–isolate, virulence declined.     

Quantitative proteomic analysis of G‐protein signalling in Stagonospora nodorum using isobaric tags for relative and absolute quantification

The G protein α‐subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2‐D LC MALDI‐MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild‐type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild‐type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up‐regulated in the gna1 strain while mannitol 1‐phosphate dehydrogenase was down‐regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short‐chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.     

In vitro screening for resistance against Septoria nodorum blotch in wheat

This study was carried out to develop an in vitro test for the identification of genotypes resistant to Septoria nodorum blotch. The basis for this project was a previous study in which a crude extract of S. nodorum was used as a selective agent (Keller et al. 1994). It was possible to distinguish resistant and susceptible cultivars in an in vitro test with zygotic embryos. In our project we wanted to test whether this in vitro test can also be used to detect resistant and susceptible genotypes in early segregating populations. Specific crosses between eight winter wheat lines showing contrasting resistance reaction for S. nodorum blotch on leaves and ears were made. The resistance level of both leaf and ear was evaluated after artificial inoculation in the field for the parental lines, the F₁ progenies, as well as for segregating F₃ and F₄ populations. In addition, this plant material was tested in vitro using methods similar to those described by Keller et al. (1994), i.e. culturing immature zygotic embryos and mature seeds on selective media. A good agreement between in vitro screening and field resistance on the ear was found for the parental lines, the F₁ and F₄ generation but not for the F₃ generations. This leads to the conclusion that the in vitro screening might be integrated into wheat breeding programs. Populations showing a high susceptibility to the pathogen metabolites in vitro could be discarded. Another promising implementation for wheat breeding would be the screening of advanced breeding material or candidate partners in a crossing program for resistance on the ear. However, the in vitro screening is not precise enough to select single plants in early segregating populations. Received: 18 January 1999 / Accepted: 30 April 1999     

Disease progress of non-specialised fungal pathogens in intraspecific mixed stands of cereal cultivars. II. Field experiments

Epidemic development of Septoria nodorum was examined in pure stands and mixtures of two spring wheat cultivars, Kolibri and Maris Butler. In mixtures, disease development was reduced almost to that of the more resistant pure stand (Maris Butler). Disease levels, however, were low and no significant differences in grain yield per head were demonstrated. Disease development was further investigated for the host-pathogen combinations winter wheat/S. nodorum and winter barley/Rhynchosporium secalis. With both combinations the mixed stands again reduced disease levels to almost that of the more resistant pure stands. In general, mixed stands were more effective against R. secalis, although, with S. nodorum, disease levels were low. The yield response in mixed stands differed for the host cultivars and was not significantly changed by the presence of disease. The complexity of analysing such situations and the implications of these findings for mixtures of cultivars differing in resistance to non-specialised pathogens are discussed.