Pyrenophora teres: profile of an increasingly damaging barley pathogen

Pyrenophora teres, causal agent of net blotch of barley, exists in two forms, designated P. teres f. teres and P. teres f. maculata, which induce net form net blotch (NFNB) and spot form net blotch (SFNB), respectively. Significantly more work has been performed on the net form than on the spot form although recent activity in spot form research has increased because of epidemics of SFNB in barley-producing regions. Genetic studies have demonstrated that NFNB resistance in barley is present in both dominant and recessive forms, and that resistance/susceptibility to both forms can be conferred by major genes, although minor quantitative trait loci have also been identified. Early work on the virulence of the pathogen showed toxin effector production to be important in disease induction by both forms of pathogen. Since then, several laboratories have investigated effectors of virulence and avirulence, and both forms are complex in their interaction with the host. Here, we assemble recent information from the literature that describes both forms of this important pathogen and includes reports describing the host-pathogen interaction with barley. We also include preliminary findings from a genome sequence survey. TAXONOMY: Pyrenophora teres Drechs. Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Dothideomycete; Order Pleosporales; Family Pleosporaceae; Genus Pyrenophora, form teres and form maculata. IDENTIFICATION: To date, no clear morphological or life cycle differences between the two forms of P. teres have been identified, and therefore they are described collectively. Towards the end of the growing season, the fungus produces dark, globosely shaped pseudothecia, about 1-2mm in diameter, on barley. Ascospores measuring 18-28μm × 43-61μm are light brown and ellipsoidal and often have three to four transverse septa and one or two longitudinal septa in the median cells. Conidiophores usually arise singly or in groups of two or three and are lightly swollen at the base. Conidia measuring 30-174μm × 15-23μm are smoothly cylindrical and straight, round at both ends, subhyaline to yellowish brown, often with four to six pseudosepta. Morphologically, P. teres f. teres and P. teres f. maculata are indistinguishable. HOST RANGE: Comprehensive work on the host range of P. teres f. teres has been performed; however, little information on the host range of P. teres f. maculata is available. Hordeum vulgare and H. vulgare ssp. spontaneum are considered to be the primary hosts for P. teres. However, natural infection by P. teres has been observed in other wild Hordeum species and related species from the genera Bromus, Avena and Triticum, including H. marinum, H. murinum, H. brachyantherum, H. distichon, H. hystrix, B. diandrus, A. fatua, A. sativa and T. aestivum (Shipton et al., 1973, Rev. Plant Pathol. 52:269-290). In artificial inoculation experiments under field conditions, P. teres f. teres has been shown to infect a wide range of gramineous species in the genera Agropyron, Brachypodium, Elymus, Cynodon, Deschampsia, Hordelymus and Stipa (Brown et al., 1993, Plant Dis. 77:942-947). Additionally, 43 gramineous species were used in a growth chamber study and at least one of the P. teres f. teres isolates used was able to infect 28 of the 43 species tested. However, of these 28 species, 14 exhibited weak type 1 or 2 reactions on the NFNB 1-10 scale (Tekauz, 1985). These reaction types are small pin-point lesions and could possibly be interpreted as nonhost reactions. In addition, the P. teres f. teres host range was investigated under field conditions by artificially inoculating 95 gramineous species with naturally infected barley straw. Pyrenophora teres f. teres was re-isolated from 65 of the species when infected leaves of adult plants were incubated on nutrient agar plates; however, other than Hordeum species, only two of the 65 host species exhibited moderately susceptible or susceptible field reaction types, with most species showing small dark necrotic lesions indicative of a highly resistant response to P. teres f. teres. Although these wild species have the potential to be alternative hosts, the high level of resistance identified for most of the species makes their role as a source of primary inoculum questionable. DISEASE SYMPTOMS: Two types of symptom are caused by P. teres. These are net-type lesions caused by P. teres f. teres and spot-type lesions caused by P. teres f. maculata. The net-like symptom, for which the disease was originally named, has characteristic narrow, dark-brown, longitudinal and transverse striations on infected leaves. The spot form symptom consists of dark-brown, circular to elliptical lesions surrounded by a chlorotic or necrotic halo of varying width.     

Mapping of major spot-type and net-type net-blotch resistance genes in the Ethiopian barley line CI 9819.

Net blotch of barley (Hordeum vulgare L.), caused by the fungal phytopathogen Pyrenophora teres Drechs. f. teres Smedeg., constitutes one of the most serious constraints to barley production worldwide. Two forms of the disease, the net form, caused by P. teres f. teres, and the spot form, caused by P. teres f. maculata, are differentiated by the type of symptoms on leaves. Several barley lines with major gene resistance to net blotch have been identified. Earlier, one of these was mapped in the Rolfi x CI 9819 cross to barley chromosome 6H, using a mixture of 4 Finnish isolates of P. teres f. teres. In this study, we used the same barley progeny to map resistance to 4 spot-type isolates and 4 net-type isolates of P. teres. With all net-type isolates, a major resistance gene was located on chromosome 6H, in the same position as described previously, explaining up to 88% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt5. Several minor resistance genes were located on chromosomes 1H, 2H, 3H, 5H, and 7H. These minor genes were not genuinely isolate-specific, but their effect varied among isolates and experiments. When the spot-type isolates were used for infection, a major isolate-specific resistance gene was located on chromosome 5H, close to microsatellite marker HVLEU, explaining up to 84% of the phenotypic variation in infection response in the progeny. We designate this gene Rpt6. No minor gene effects were detected in spot-type isolates. The Ethiopian 2-rowed barley line CI 9819 thus carries at least 2 independent major genes for net-blotch resistance: Rpt5, active against net-type isolates; and Rpt6, active against specific spot-type isolates.     

Identification and chromosomal location of major genes for resistance to Pyrenophora teres in a doubled-haploid barley population.

Net blotch, caused by Pyrenophora teres, is one of the most economically important diseases of barley worldwide. Here, we used a barley doubled-haploid population derived from the lines SM89010 and Q21861 to identify major quantitative trait loci (QTLs) associated with seedling resistance to P. teres f. teres (net-type net blotch (NTNB)) and P. teres f. maculata (spot-type net blotch (STNB)). A map consisting of simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers was used to identify chromosome locations of resistance loci. Major QTLs for NTNB and STNB resistance were located on chromosomes 6H and 4H, respectively. The 6H locus (NTNB) accounted for as much as 89% of the disease variation, whereas the 4H locus (STNB resistance) accounted for 64%. The markers closely linked to the resistance gene loci will be useful for marker-assisted selection.     

Population genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis>Drechs. the causal agent of net blotch in Sardinian landraces of barley (<Emphasis Type="Italic">Hordeum vulgare L.</Emphasis>)

Monoconidial cultures of Pyrenophora teres, the causal agent of barley net blotch, were isolated from leaves collected from six populations of the barley landrace "S'orgiu sardu" growing in five agro-ecological areas of Sardinia, Italy, and genotyped using AFLPs. The 150 isolates were from lesions of either the "net form" (P. teres f. sp. teres) or the "spot form" (P. teres f. sp. maculata) of the disease. Of 121 AFLP markers, 42%, were polymorphic. Cluster analysis resolved the isolates into two strongly divergent groups (F(ST) = 0.79), corresponding to the net (45% of the isolates) and the spot (55% of the isolates) forms (designated the NFR and SFR groups, respectively). The absence of intermediate genotypes and the low number of shared markers between the two groups indicated that hybridization between the two formae is rare or absent under the field condition of Sardinia. Five of the barley populations hosted both forms but in different proportions. The SFR populations were similar in overall polymorphism to the NFR populations. However, compared to the SFR form, the NFR occurred in all fields sampled and showed a higher population divergence (F(ST) = 0.43 versus F(ST) = 0.09 with all isolates; F(ST) = 0.37 versus F(ST) = 0.06 with clone corrected samples) probably due to a lower migration rate. AFLP fingerprints resolved 117 distinct genotypes among the 150 isolates sampled (78%), 87% in SFR and 68% in NFR isolates. Although the absolute numbers may be a function of the number of AFLP markers assayed, the relative difference suggests that clonality is more prevalent among the NFR isolates (with 11 of 46 haplotypes observed more than once), compared with SFR isolates (7 of 71 haplotypes). Both digenic and multilocus linkage disequilibrium analyses suggested that sexual reproduction occurs at significant levels within the NFR and SFR populations, and that the relative contribution of sexual and asexual reproduction varies among different environments.     

Quantification of Pyrenophora teres in infected barley leaves using real-time PCR

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R(2)=0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.     

Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres and frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces.

Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.     

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).     

Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota.

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

Homologs of ToxB, a host-selective toxin gene from Pyrenophora tritici-repentis, are present in the genome of sister-species Pyrenophora bromi and other members of the Ascomycota

Pyrenophora tritici-repentis requires the production of host-selective toxins (HSTs) to cause the disease tan spot of wheat, including Ptr ToxA, Ptr ToxB, and Ptr ToxC. Pyrenophora bromi, the species most closely related to P. tritici-repentis, is the causal agent of brown leaf spot of bromegrass. Because of the relatedness of P. bromi and P. tritici-repentis, we investigated the possibility that P. bromi contains sequences homologous to ToxA and/or ToxB, the products of which may be involved in its interaction with bromegrass. Multiplex polymerase chain reaction (PCR) revealed the presence of ToxB-like sequences in P. bromi and high-fidelity PCR was used to clone several of these loci, which were subsequently confirmed to be homologous to ToxB. Additionally, Southern analysis revealed ToxB from P. bromi to have a multicopy nature similar to ToxB from P. tritici-repentis. A combination of phylogenetic and Southern analyses revealed that the distribution of ToxB extends further into the Pleosporaceae, and a search of available fungal genomes identified a distant putative homolog in Magnaporthe grisea, causal agent of rice blast. Thus, unlike most described HSTs, ToxB homologs are present across a broad range of plant pathogenic ascomycetes, suggesting that it may have arose in an early ancestor of the Ascomycota.     

Genetic control of virulence of Pyrenophora teres drechs, the causative agent of net blotch in barley

The genetic control of virulence was studied in four isolates of the fungus Pyrenophora teres f. teres, originating from various geographic regions in experiments with nine barley accessions, possessing known resistance genes. Experiments were performed with the ascospore progeny of two crosses. The results of segregation for virulence in the progeny of direct crosses were confirmed by analysis of backcrosses and sib crosses. One to four genes for avirulence toward various barley genotypes were found in the isolates under study. It is suggested that dominant suppressor genes are involved in the genetic control of avirulence toward four barley genotypes.     

Sensitivity of Resistance to Net Blotch in Barley

The aim of this study was to demonstrate various methods of analysing terminal net blotch, Pyrenophora teres Drechs. f. teres Smedeg., severity data from 15 spring barleys, Hordeum vulgare L., grown in Finnish official variety trials in five environments. The analyses have been developed and used principally by plant breeders for assessing crop yield, but lend themselves to use by plant pathologists. Pyrenophora teres is the major barley phytopathogen in Finland and improved resistance to it is sought. Joint regression analysis (JRA) and an additive main effects and multiplicative interaction (AMMI) model were used to investigate the data. Statistically significant genotype by environment (GE) interaction for resistance was indicated, and this included qualitative (crossover) interactions among genotypes over environments. A stable, non-sensitive, response to net blotch over environments, combined with a low mean score for terminal severity of the disease characterized the six-row barley 'Thule' which showed statistically significant crossover interaction only with 'Tyra'. 'Kustaa' exhibited the lowest mean terminal net blotch severity, but was relatively sensitive to net blotch. 'Arve' exhibited severe terminal net blotch in all environments, was relatively sensitive to environment and exhibited no crossover interaction with other genotypes. AMMI analysis appeared to represent a useful method for analysing these disease severity data, facilitating the selection of useful sources of resistance. Plots of AMMI-adjusted mean net blotch severities against first principal component axis (PCA) scores were informative for differentiating genotype response over environments, and are therefore potentially useful to plant pathologists and barley breeders seeking to gauge and subsequently improve the resistance status of barley to net blotch.     

A biological and molecular characterization of some Egyptian barley genotypes which are resistant to net blotch disease

A survey for resistance against net blotch disease (caused by Pyrenophora teres) was performed on some Egyptian barley landraces and some selected resistance and susceptible standard German barley genotypes. The results indicated that most of the Egyptian barley landraces are extremely resistant to the disease. Molecular analysis using RAPD and AFLP showed unique banding profiles for the different genotypes, and specific AFLP markers for the Egyptian genotypes were identified. The effectiveness of RAPD and AFLP for identifying different barley genotypes of different origins and with different reactions against P. teres was discussed. The results of the biological evaluation and molecular characterization done in this study can be seen as the starting point needed to identify the valuable net blotch resistant Egyptian barley germplasm at both the phenotype and genotype levels and draw the attention of breeders and banks of natural plant genetic resources towards this valuable yet neglected germplasm.     

Inhibition of efflux transporter-mediated fungicide resistance in Pyrenophora tritici-repentis by a derivative of 4'-hydroxyflavone and enhancement of fungicide activity

Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.     

Production of a Chlorosis-Inducing, Host-Specific, Low-Molecular Weight Toxin by Isolates of Pyrenophora tritici-repentis Cause of Tan Spot of Wheat

An extraction procedure was developed to isolate toxin from cell-free culture filtrates of Pyrenophora tritici-repentis, cause of tan spot disease of wheat (Triticum aestivum). Designated Ptrtoxin, this low-molecular weight toxin (MW 800–1800) induces the characteristic chlororis associated with tan spot disease when inoculated to healthy wheat leaves and differentially inhibits the elongation of wheat seedling coleoptiles. The sensitivity of seven wheat, one barley, and non-host (corn) cultivars to Ptr-toxin corresponds with reported field reactions to the pathogen. Seven isolates of P. triticirepentis from wheat in Oklahoma and four from smooth bromegrass Bromus inermis, an alternative host of P. tritici-repentis), in North Dakota were evaluated for production of Ptr-toxin in vitro. Results indicate that Ptr-toxin may be involved in the expression of disease symptoms in tan spot of wheat, but is not the sole determinant of P. tritici-repentis pathogenicity. Instead, toxin production may contribute to the virulence of individual isolates of the pathogen. Isolates from the alternative host, smooth bromegrass, appear at least as toxigenic as those from wheat, with the exception of one which appeared more toxigenic than four of the wheat isolates tested. Possible relationships of Ptr-toxin to high-molecular weight toxins reported to be produced by P. triticirepentis in vitro (Ptr-necrosis-toxin) are not understood.     

RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.     

Sexual reproduction and soil tillage effects on virulence of Pyrenophora teres in Finland

Knowledge of the virulence of a pathogen population and recognition of the risks of changes in the virulence spectrum are essential in breeding crops for disease resistance. Sexual recombination in a pathogen increases the level of genotypic diversity and can influence the virulence spectrum. This study aimed to determine how sexual recombination can change virulence of the barley pathogen Pyrenophora teres and whether the barley cultivation system, no‐tillage or normal tillage, influences P. teres virulence. The inheritance of avirulence/virulence in P. teres following sexual reproduction was studied in three artificially created pathogen populations. The first was a product of crossing two net forms of the pathogen, and the second and the third were products of crossing net and spot forms. None of the progeny generated resembled the parents exactly. The average similarity of the progeny isolates of the net by net cross with the parental type, based on avirulence/virulence tests, was 92%. That for net and spot form progenies was 58% in comparison with the net form parents and 73% with the spot form parents. The virulence reactions of the progeny isolates did not correlate with morphological traits of the isolates: growth rate on agar, spore production, spore width, spore length and numbers of septa per conidium. To study the effect of the barley cultivation method on P. teres virulence, 313 single‐spore cultures were obtained from barley fields. Two hundred and seventy‐six of the isolates represented the spot form and 37 represented the net form of P. teres. No association was established between the tillage method and virulence for either the net form or the spot form isolates.     

Pyrenophora teres,an agent causing wheat leaf spot

In 2007–2008, the barley net blotch agent Pyrenophora teres was found to infect spring wheat in northwestern Russia, causing symptoms similar to wheat tan spot caused by P. tritici-repentis. The frequency of occurrence of P. teres on spring wheat cultivars was 12–29%. P. teres isolates were more virulent to some wheat cultivars than P. tritici-repentis ones. P. teres was not found on wheat in the south of Russia (Krasnodar krai, Dagestan).