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.     

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.     

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.     

Comparative virulence of Pyrenophora teres f. teres from Syria and Tunisia and screening for resistance sources in barley: implications for breeding

Aims: The aim of this study is to investigate the pathogenic diversity and virulence groups among Pyrenophora teres f. teres isolates, sampled from Syria and Tunisia, and to identify the most effective source of resistance in barley that could be used in breeding programmes to control net blotch in both countries. Methods and Results: One hundred and four isolates of P. teres f. teres were collected from barley in different agroecological zones of Tunisia and Syria. Their virulence was evaluated using 14 barley genotypes as differential hosts. The upgma clustering identified high pathogenic variability; the isolates were clustered onto 20 pathotypes that were sheltered under three virulence groups, with high, intermediate and low disease scores. According to susceptibility/resistance frequencies and mean disease ratings, CI05401 cultivar ranked as the best differential when inoculated with the Syrian isolates. However, CI09214 cultivar was classified as the best effective source of resistance in Tunisia. Conclusions: All P. teres f. teres isolates were differentially pathogenic. CI09214 and CI05401 cultivars were released as the most effective sources of resistance in Syria and Tunisia. Significance and Impact of the Study: National and international barley breeding programmes that seek to develop resistance against P. teres f. teres in barley should strongly benefit from this study. This resistance cannot be achieved without the proper knowledge of the pathogen virulence spectrum and the sources of host resistance.     

Genetic mapping of Pyrenophora teres f. teres genes conferring avirulence on barley

A Pyrenophora teres f. teres cross between isolates 0-1 and 15A was used to evaluate the genetics of avirulence associated with barley lines Canadian Lake Shore (CLS), Tifang, and Prato. 15A is avirulent on Tifang and CLS, but virulent on Prato. Conversely, 0-1 is avirulent on Prato, but virulent on Tifang and CLS. Avirulence:virulence on Tifang and CLS segregated 1:1, whereas avirulence:virulence on Prato segregated 3:1. An AFLP-based linkage map was constructed and used to identify a single locus derived from 15A (AvrHar) conferring avirulence to Tifang and CLS. Virulence on Prato was conferred by two epistatic genes (AvrPra1 and AvrPra2). AvrPra2 co-segregated with AvrHar, but the two genes from opposite parents conferred opposite reactions. This work provides the foundation for the isolation of these avirulence genes.     

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.     

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.     

Interaction between Rhynchosporium secalis and Pyrenophora teres in the Field and Identification of Genotypes with Double Resistance in a Doubled-haploid Barley Population

Net blotch [Pyrenophora teres (Died.) Drechsl.] and scald [Rhynchosporium secalis (Oudem.) J.J. Davis] are the two most important foliar diseases of barley (Hordeum vulgare L.) in Tunisia. The use of cultivars with double resistance is the most effective method in controlling both diseases. A doubled‐haploid barley population derived from Tunisian cultivars was evaluated to both net blotch and scald during two growing seasons in the field. Mass disease index (MDI), area under the disease progress curve (AUDPC) and apparent infection type (r) were used to assess disease reaction. MDI of net blotch and scald reached up to 65% and 90% respectively. Least significant difference (LSD) test and comparison of the reaction of the doubled haploid (DH) lines to the overall population mean value were efficient in identifying lines with double resistance to both diseases. From the 59 DH lines screened, lines 21, 33, 37, 46 and 47 showed the best level of adult plant resistance to both diseases and may be used in a breeding program for diseases resistance. Interactions between R. secalis and P. teres were investigated at the level of the whole plant under variable epidemic conditions. Under low epidemic conditions, net blotch and scald developments were usually independent, but positively associated for tolerant lines for both diseases. Under high epidemic conditions, competition effects were obtained for susceptible and resistant genotypes. This competition seems to be an exploitation competition that is associated with decreasing resource availability as it occurs only with high levels of infestation or/and when susceptible lines are infected. This study demonstrates the variability of net blotch and scald interaction with epiphytotic conditions and group of resistance.     

Necrotrophic effector‐triggered susceptibility (NETS) underlies the barley–Pyrenophora teres f. teres interaction specific to chromosome 6H

Barley net form net blotch (NFNB), caused by the necrotrophic fungus Pyrenophora teres f. teres, is a destructive foliar disease in barley‐growing regions worldwide. Little is known about the genetic and molecular basis of this pathosystem. Here, we identified a small secreted proteinaceous necrotrophic effector (NE), designated PttNE1, from intercellular wash fluids of the susceptible barley line Hector after inoculation with P. teres f. teres isolate 0–1. Using a barley recombinant inbred line (RIL) population developed from a cross between the sensitive/susceptible line Hector and the insensitive/resistant line NDB 112 (HN population), sensitivity to PttNE1, which we have named SPN1, mapped to a common resistance/susceptibility region on barley chromosome 6H. PttNE1–SPN1 interaction accounted for 31% of the disease variation when the HN population was inoculated with the 0–1 isolate. Strong accumulation of hydrogen peroxide and increased levels of electrolyte leakage were associated with the susceptible reaction, but not the resistant reaction. In addition, the HN RIL population was evaluated for its reactions to 10 geographically diverse P. teres f. teres isolates. Quantitative trait locus (QTL) mapping led to the identification of at least 10 genomic regions associated with disease, with chromosomes 3H and 6H harbouring major QTLs for resistance/susceptibility. SPN1 was associated with all the 6H QTLs, except one. Collectively, this information indicates that the barley–P. teres f. teres pathosystem follows, at least partially, an NE‐triggered susceptibility (NETS) model that has been described in other necrotrophic fungal disease systems, especially in the Dothideomycete class of fungi.     

Increased Levels of Cytokinins in Barley Leaves Having the Systemic Acquired Resistance to Bipolaris sorokiniana (Sacc.) Shoemaker

The role of endogenously induced higher level of cytokinins and exogenously applied kinetin in relation to the development of barley leaf spot caused by Bipolaris sorokiniana (Sacc.) Shoemaker (syn. Helminthosporium sativum Pammel, King and Bakke) was studied. Spraying barley leaves with kinetin suppressed the number and the size of necrotic spots caused by the fungus. Inoculation of the lower leaves of barley by a spore suspension of the fungus B. sorokiniana induced resistance on the upper leaves against a subsequent challenge inoculation by the same pathogen 10 days later. An increase in the level of cytokinins was observed in these resistant leaves. Elevated levels of cytokinins may cause a type of juvenility in leaf tissues. The juvenile state could be in a causal relationship with the suppression of necrotic spots caused by the fungus.     

Toxin production in Pyrenophora teres, the ascomycete causing the net-spot blotch disease of barley (Hordeum vulgare L.)

Toxin production in a large number of Pyrenophora teres isolates have been investigated. During fungal growth, the pH of the medium decreases from 6.5 to about 3.0. Aspergillomarasmine A is the major toxin excreted into the culture medium. Nonenzymatic acid-catalyzed conversion of aspergillomarasmine A to anhydroaspergillomarasmine A proceeds at low pH and is prevented by repeated titration of the culture medium to pH 6.5. The four possible stereoisomers of N-(2-amino-2-carboxyethyl)aspartic acid have been chemically synthesized and their absolute configuration determined. From circular dichroism and NMR spectroscopy and by measurements of specific optical rotation, the LL-form is identified as the stereoisomer produced by P. teres. Biosynthetic experiments using radioisotopes demonstrate that the LL-isomer of N-(2-amino-2-carboxyethyl) aspartic acid is a direct precursor of aspergillomarasmine A. Consequently, L-configuration is assigned to the two corresponding asymmetric carbon atoms of aspergillomarasmine A. This is in contrast to earlier reports which had indicated D configuration. The phytotoxicity of anhydroaspergillomarasmine A is comparable with that of L-aspartic acid, whereas LL-N-(2-amino-2-carboxyethyl)aspartic acid exerts strong phytotoxicity in the bioassays as shown previously for aspergillomarasmine A. The amount of LL-N-(2-amino-2-carboxyethyl)aspartic acid which accumulates in the P. teres cultures is low, indicating that aspergillomarasmine A is the toxin which plays the major role in the pathological changes associated with the barley net-spot blotch disease.     

Changes in specific activities of peroxidase, chitinase and phenylalanine ammonia-lyase and phenolic content in cucumber leaves inoculated with Podosphaera fusca, the causal agent of powdery mildew

In this investigation, the effects of powdery mildew disease [caused by Podosphaera fusca (syn. Sphaerotheca fuliginea)] on specific activities of several defense-related enzymes and phenolic content were studied in cucumber leaves. Spore suspension of the fungus was sprayed on cucumber (cv. Super Dominus) plants in greenhouse and leaves from both inoculated and non-inoculated control plants were sampled at 0, 24, 48, 72 and 144 hours after inoculation (HAI). Spore germination and tissue colonization of P. fusca were microscopically studied on the inoculated surface of leaf samples. Further, Phenolic content (PHE) and specific activities of peroxidase (POX), chitinase (CHI) and phenylalanine ammonia-lyase (PAL) were spectrophotometrically measured in leaf extracts. Time-course of disease progress on the leaf surface showed that maximum spore germination occurred within 24 HAI and host penetration and disease development process began during 24-48 HAI. Evaluation of enzyme activities showed that POX specific activity in inoculated plants significantly increased at 72 HAI onwards and reached 2.5 times of that of control at 144 HAI. CHI specific activity showed a transient reduction in inoculated plants between 48-72 HAI and thereafter increased significantly in relation to control. PAL specific activity in inoculated plants was not significantly different from that of control. PHE in inoculated plants showed a significant increase compared to control at 48 HAI and thereafter. Comparison of time-course of disease progress with changes in enzyme activities indicated that POX activity had an increasing trend during disease progress whereas CHI activity showed a transient decrease at the early stages and then increased during the later stages of infection: PAL activity did not show any changes during the infection and PHE increased at the early stages of infection process and remained constant at rest of the time.     

Pyrenophora teres: Population structure,virulence and aggressiveness in Southern Russia

The barley net blotch agent Pyrenophora teres (Died) Drechs. is one of the dominant fungal pathogens in agricultural crops worldwide. Here we aim to study the aggressiveness and virulence of P. teres populations collected at different ontogenesis stages (BBCH 30 and BBCH 47) from winter barley cultivars of various resistance types: moderately resistant, moderately susceptible and highly susceptible. We observed a direct proportional relationship between cultivar resistance and the aggressiveness of P. teres populations collected in both growth phases of the host plant. The isolates collected at an early stage of host plant development have a large difference in aggressiveness criteria: colony growth rate, sporulation intensity, latency period, plant damage degree, and the number of identified races. At the BBCH 30 growth stage, the growth rate of fungus colonies selected from a resistant cultivar is 1.2 times higher than that of a susceptible cultivar. The growth rate of colonies selected from resistant and susceptible cultivars in the earlier BBCH 30 stage is 1.04 times higher than the growth rate of colonies selected from the later phase. The sporulation intensity of fungal populations selected from a resistant cultivar is higher than that of populations selected from a susceptible cultivar (for BBCH 30–5.4 times, for BBCH 47–4.0 times); and it is 1.3 times higher in an earlier phase of plant development. Correlation between colony growth rate and spore formation rate in the BBCH 30 is r = 0.4. A high correlation level (r = 0.9) and notable difference between the variants were revealed when studying the duration of the latent period. The average value of plant damage by the P. teres from resistant cultivar is 4 times higher than from the susceptible cultivar in the BBCH 30 stage; and 12 times – in the BBCH 47 stage. There is a moderate negative correlation between the plant damage degree and the number of races identified from the fungal population, r = ?0.59 for the BBCH 30, r = ?0.8 for the BBCH 47. The number of races identified from P. teres populations collected in the late phase of plant growth was one third less. Our study helped to acquire new knowledge about intrapopulation processes under the influence of various factors – plant growth stage and cultivar genotype. The results obtained are the basis for the development of adaptive-integrated techniques for managing populations of the hemibiotrophic pathogen, barley 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.     

Influence of Temperature on the Virulence of Two Races of Meloidogyne chitwoodi on Wheat and Barley

Races of the Columbia root-knot nematode, Meloidogyne chitzooodi, from Idaho (R1) and Utah (R2) suppressed (P < 0.05) tillering of Dusty winter wheat, Fielder spring wheat, Luther winter barley, and Steptoe spring barley at 15-30 C. Nematode inoculum density was negatively correlated with tillering (r = -0.79). Inoculum densities of both nematode races were negatively correlated with heads per plant (r = -0.83), head length (r = -0.87), and head dry weight (r = 0.73) of Fielder spring wheat and Steptoe spring barley at all temperatures; the greatest growth restrictions occurred at Pi 20 eggs/cm³ soil. Both nematode races were most damaging at 25-30 C. Fielder spring wheat and Steptoe spring barley inoculated with R2 produced fewer heads than R1 when inoculated at 15 C, whereas the same cultivars inoculated with R1 produced fewer heads than R2 at 30 C. No differences were observed between root growth of winter and spring wheat or between winter and spring barley. Nematode reproduction was positively correlated to temperature (r = 0.87) and negatively correlated with inoculum density (r = -0.86). Reproductive rates were greatest with Pi = 2 eggs/cm³ soil at 25 C and lowest with Pi = 20 eggs/cm³ soil at 15 C for both nematode races.     

An analysis of rain-mediated dispersal of Drechslera teres conidia in field plots of spring barley

In two separate trials rain-mediated dispersal of Drechslera teres conidia was observed in field plots of cv. Beatrice spring barley. Within the crop spore number was greater towards the base reflecting both the downward movement of conidia and the greater availability of spores on older leaves. From the edge of the crop half as many conidia were trapped at 100 cm as at 25 cm. In both trials the cumulative spore catch total lagged behind the increase in net blotch infection levels on the top two leaves. The number of spores sampled appeared closely related to rainfall intensity.