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Cereals are a significant agricultural product group with the highest cultivation area and production in Turkey. Among these cereals, barley is an important cereal used in human and animal nutrition, and the most widelycultivated plant after wheat. There are biotic and abiotic factors which affect the yield and quality parameters of barley. Of the biotic factors, barley leaf stripe (agent: Pyrenophora graminea (anamorph: Drechslera graminea) is an important fungal disease. Infections can occur in diseased seeds and in the presence of suitable environmental conditions. Hence, the use of disease-free seeds and resistant cultivars (cvs.) against this pathogen are crucial. With this in mind, this study was carried out with a highly virulent isolate of Pyrenophora graminea, obtained from the Disease and Pest Resistance Unit culture collection in 57 registered barley cultivars in the greenhouses of the Central Research Institute of Field Crops (TARM) in 2021. The experiments were conducted in 3 replications in a randomized block design. Of 57 registered barley cultivars 62%, 15% and 36% of these barley cultivars expressed resistance, moderate resistance, and susceptible reactions, respectively. As a result of the analysis of variance performed on the results obtained, it was concluded that the difference between the mean values of 57 varieties was statistically significant at 1% level (P<0.01). [ABSTRACT FROM AUTHOR]

This study aims at monitoring the dynamics of the occurrence and evolution of the attack of some pathogens to barley, among which we mention: mildew (Blummeria graminis f.sp. hordei), leaf stripe (Pyrenophora graminea) and barley's rust (Puccinia hordei). Also, the influence of applying these fungicides on the harvest, as well as of the number of treatments/ha as compared to the untreated control variant, has been monitored. For this study, an experiment with 6 treatment variants was created, being used the following phytosanitary products: EVALIA (azoxystrobin 250 g/l), RETENGO (200 g/l pyraclostrobin) and NATIVO PRO 325 SL (prothioconazole 175 g/l + trifloxystrobin 150 g/l). The treatment variants were the following: V1-RETENGO 0.8 l/ha, 1 treatment was applied on 13th April + 1 treatment on 17th May; V2 - EVALIA 0.75 l/ha, 1 treatment was applied on 13th April +1 treatment on 17th May; V3-NATIVO PRO 0.6l/ha, 1 treatment was applied on 13th April + 1 treatment on 17th May; V4- RETENGO 0.8 l/ha, 1 treatment was applied on 22nd May, V5- EVALIA 1.0 l/ha, 1 treatment was applied on 22nd May, V6- NATIVO 0.7 l/ha, 1 treatment was applied on 22nd May, V7 - Untreated Control Variant. The experiment was placed in Latin square, the 7 variants being placed in 7 repetitions. The year 2022 was a year where the spring and the beginning of summer were very dry, totally unfavorable to pathogen attacks. The experiment was not irrigated. Among the pathogens monitored, very weak attacks by the fungus Pyrenophora graminea, which produces, in barley, the disease known as leaf stripe, were observed. This led to very uniform yields in the variants studied. Basically, no significant yield differences occurred in the climate conditions of 2022 between the untreated control variant and the variants that were treated with fungicides. The variant's yields were: V1 - 5,276 to/ha, V2 - 5,292 to/ha, V3 - 5,451 to/ha, V4 - 5,184 to/ha, V5 - 5,601 to/ha, V6 - 5,585 to/ha and V7 - 5,508 to/ha. [ABSTRACT FROM AUTHOR]

Barley leaf stripe, caused by Pyrenophora graminea, is an essential systemic seed-borne disease in barley worldwide. Barley is a major cereal crop in the Qinghai–Tibet Plateau, and barley production has been threatened by leaf stripe in this region, particularly in organic farming regions. Detecting the pathogen in infected barley seeds is crucial for managing barley leaf stripe. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the pathogen based on primers designed based on the sequence of the pig 14 gene (GenBank: AJ277800) of P. graminea. The optimal concentrations of MgSO4, dNTPs, and enzymes in the LAMP reaction system were established as 10.0 mM, 1.0 mM, and 8 U in a 25 μL reaction volume, respectively. The established LAMP methods for detecting P. graminea were optimally performed at 63 °C for 70 min with high reliability. The minimum detection limit was 1 × 10−2 ng·μL−1 in the 25 μL reaction system. The specificity of LAMP for P. graminea was validated with eight fungal species. All DNA extracts from P. graminea-infected barley seeds with incubation, intact, and smashed treatments were applied in LAMP and confirmed to enable the detection of the pathogen. The LAMP assay in this study could facilitate the detection of P. graminea in barley seeds onsite, provide information for seed health certificates, and help decide on seed treatment in leaf stripe management. [ABSTRACT FROM AUTHOR]

Leaf stripe is a common, but major infectious disease of barley, severely affecting the yield and quality. However, only a few genes have been identified by conventional gene mapping. Gene family analysis has become a fast and efficient strategy for gene discovery. Studies demonstrated that Argonaute (AGO) proteins play an important role in plant disease resistance. Thus, we obtained nine HvAGO genes via mRNA sequencing before and after a Pyrenophora graminea infection of a disease-resistant variety "Kunlun 14" and a susceptible variety "Z1141". We analysed the physicochemical characteristics, gene structures, and motifs of the HvAGO gene sequences and found that these proteins were divided into four clusters by evolutionary distance. There was high consistency in the number of exons, size, and the number and type of motifs in the different clusters. Based on protein phylogenetics, they could be divided into three branches. A collinearity analysis of Tibetan hull-less barley and Arabidopsis thaliana, rice, and maize showed that four genes were collinear with respect to the other three species. The qRT-PCR showed the expression levels of HvAGO1, HvAGO2 and HvAGO4 were significantly increased after infection with Pyrenophora graminea. These three members of the AGO gene family are, thus, speculated to play an important role in barley leaf stripe resistance. The results provide reference for the application of HvAGO genes in the leaf stripe control and the exploration of disease resistance genes in other crops.

Barley leaf stripe is an important disease caused by Pyenophora graminea that affects barley yields in the world. Ascorbic acid (AsA) interacts with key elements of a complex network orchestrating plant defense mechanisms, thereby influencing the outcome of plant-pathogen interaction. Myo-inositol oxygenase (MIOX) is a pivotal enzyme involved in plants development and environmental stimuli. However, MIOX has described functions in plants but has not been characterized in fungi. In this study, we characterized the Pgmiox gene in P. graminea pathogenesis through annotated on the metabolic pathway of ascorbic acid aldehyde. Our analysis suggested that the Pgmiox protein had a typical conserved MIOX domain. Multiple alignment analysis indicated that the P. graminea MIOX orthologue clustered with MIOX proteins of Pyrenophora species. RNA interference successfully reduced transcript abundance of Pgmiox in six transformant lines compared to wild type, and the transformants were further less virulent on the host plant barley. Transformants of Pgmiox had significant reductions in vegetative growth and pathogenicity, which had increased resistance to tebuconazole and carbendazim. In addition, Pgmiox is associated with ionic, drought, osmotic, oxidative, and heavy metal stress tolerance in P. graminea. In conclusion, our findings reveal that Pgmiox may be widely utilized by fungi to enhance pathogenesis and holds significant potential for the development of durable P. graminea resistance through genetic modifications. • We efficaciously obtained P. graminea transformants harboring silenced types of Pgmiox. • Pgmiox is required for response to osmosensitivity and fungicide tolerance of P. graminea. • Pgmiox is not only related to cell wall integrity, but also necessary for the pathogenicity in P. graminea. [ABSTRACT FROM AUTHOR]

The utilization of production systems with reduced chemical input renewed the interest in Ustilago nuda and Pyrenophora graminea. The investigations of seed fungicide treatments are more related to their efficacy than to their contribution to yield gain. The data were collected from research and development trials on fungicide efficacy against U. nuda and P. graminea conducted from 2014 to 2020 in Serbia. Partial least squares, multiple stepwise regression and best subset regression were used for statistical modeling. The total number of plants infected with U. nuda and P. graminea per plot differed significantly in the seven-year period. Shifts in the predominance of one pathogen over the other were also shown. Temperature, total rainfall and relative humidity in flowering time (p < 0.001) influenced the occurrence of both pathogens. The strongest impact on yield loss was observed for temperature in the phenological phases of leaf development (p = 0.014), temperature in flowering time (p < 0.001) and total number of plants infected with U. nuda and P. graminea per plot (p < 0.001). Our results indicated that regression models consisting of both biotic and abiotic factors were more precise in estimating regression coefficients. Neither fungicidal treatment had a stable contribution to yield gain in the seven-year period. [ABSTRACT FROM AUTHOR]

[Objective] To provide a reference for detection of barley seeds and effective prevention and control of barley stripe, this study established a detection system for barley stripe based on loop-mediated isothermal amplification (LAMP) technology. [Method] In this study, five sets of specific primers were de-signed based on the ITS-rDNA sequences of P. graminea, and one set was screened to optimize reaction temperature based on six gradients (60, 61, 62, 63, 64 and 65 °C). Then, the LAMP system for efficient detection of the pathogen was established. Its specificity was tested based on LAMP amplification of DNA of genes from P. graminea, Ustilago hordei, Ustilago nuda, Dactylobotrys graminicola, Alternaria alternata and Fusarium graminearum. The sensitivity was analyzed by testing genes at levels of 310, 31, 3. 1, 0.31, 0.031 and 0.003 1 pg/µL. At last, it was used for testing and verifying seeds of five barley varieties. [Result] The established LAMP methods for detecting P. graminea performed optimally at 63 °C for 70 min with specific primers of set 2. Under the optimal reaction conditions, this method had good specificity for P. graminea with minimum detection limit of 0.31 pg/µL. The P. graminea-carrying rates of Dulihuang, Chaiqing 1, 1141, Beiqing 9 and Kunlun 15 seeds were 68.18%, 72.73%, 100%, 77.27% and 68.18%, respectively. [Conclusion] The established LAMP method can be used to detect the P. graminea in barley seeds within 70 min at 63 µC. [ABSTRACT FROM AUTHOR]

Barley leaf stripe, caused by Pyrenophora graminea, is an essential systemic seed-borne disease in barley worldwide. Barley is a major cereal crop in the Qinghai–Tibet Plateau, and barley production has been threatened by leaf stripe in this region, particularly in organic farming regions. Detecting the pathogen in infected barley seeds is crucial for managing barley leaf stripe. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect the pathogen based on primers designed based on the sequence of the pig 14 gene (GenBank: AJ277800) of P. graminea. The optimal concentrations of MgSO4, dNTPs, and enzymes in the LAMP reaction system were established as 10.0 mM, 1.0 mM, and 8 U in a 25 μL reaction volume, respectively. The established LAMP methods for detecting P. graminea were optimally performed at 63 °C for 70 min with high reliability. The minimum detection limit was 1 × 10−2 ng·μL−1 in the 25 μL reaction system. The specificity of LAMP for P. graminea was validated with eight fungal species. All DNA extracts from P. graminea-infected barley seeds with incubation, intact, and smashed treatments were applied in LAMP and confirmed to enable the detection of the pathogen. The LAMP assay in this study could facilitate the detection of P. graminea in barley seeds onsite, provide information for seed health certificates, and help decide on seed treatment in leaf stripe management.

The seed- borne pathogen Pyrenophora graminea Ito& Kur. is the causal agent of barley leaf stripe, a destructive disease in most barley growing region in the world. In order to improve the barley species in Algeria against this disease, it is necessary to know their tolerance reaction to it. The genotype that have been tested are five parent genotypes: ACSAD, Tichedrett, Saida, Rihane, Hamra and eight populations hybrid obtained by intraspecific crosses of the parents and then sowing of F1 to F8. We compared the incidence of the isolate of Pyrenophora graminea for morphological characters (Stem height) and yield (1000-grain weight, medium yield of plant) of the studied variety. The results obtained showed the existence of variability between our thirteen genotypes of barley which had a different resistance level in the quantitative and qualitative characters.

The utilization of production systems with reduced chemical input renewed the interest in Ustilago nuda and Pyrenophora graminea. The investigations of seed fungicide treatments are more related to their efficacy than to their contribution to yield gain. The data were collected from research and development trials on fungicide efficacy against U. nuda and P. graminea conducted from 2014 to 2020 in Serbia. Partial least squares, multiple stepwise regression and best subset regression were used for statistical modeling. The total number of plants infected with U. nuda and P. graminea per plot differed significantly in the seven-year period. Shifts in the predominance of one pathogen over the other were also shown. Temperature, total rainfall and relative humidity in flowering time (p < 0.001) influenced the occurrence of both pathogens. The strongest impact on yield loss was observed for temperature in the phenological phases of leaf development (p = 0.014), temperature in flowering time (p < 0.001) and total number of plants infected with U. nuda and P. graminea per plot (p < 0.001). Our results indicated that regression models consisting of both biotic and abiotic factors were more precise in estimating regression coefficients. Neither fungicidal treatment had a stable contribution to yield gain in the seven-year period.

Wild barley (Hordeum spontaneum) and barley landraces are important sources of genetic variation for disease resistance. Thirty wild barley (H. spontaneum) genotypes and 30 barley landraces were evaluated for susceptibility to two Drechslera graminea isolates. Virulence differences were observed between the isolates, while the responses of the host genotypes to the isolates also varied. Of the H. spontaneum genotypes, 23% and 63%, respectively, were resistant to the Yozgat D. graminea isolate, and Eskişehir D. graminea isolates. On the other hand, 43% and 90% of the barley landraces were resistant to Yozgat and Eskişehir D. graminea isolates, respectively. Hordeum spontaneum genotypes 13, 24, 27, 29, 54, 86, and 91 exhibited resistance to both D. graminea isolates, while genotypes 14 and 32 showed intermediate reactions to the Yozgat isolate and resistant reactions to the Eskişehir isolate. Barley landraces 21, 37, 38, 39, 40, 73, 98, 128, 139, 153, 159,167, and 171 showed resistant reactions, and barley landrace 8 showed an intermediate reaction to both isolates. Barley landraces 3, 20, 24, 71, 101, 103, 104 and 160 exhibited intermediate responses to the Yozgat isolate and a resistant response to the Eskişehir isolate. Using resistant barley genotypes would reduce the need for pesticides for control of leaf stripe, and be an environmentally preferred strategy for disease control. The disease resistance present in wild barley and barley landraces are important for expanding the genetic basis of cultivated barley (H. vulgare). The resistant and intermediate genotypes identified in this study could be used as resistance sources in barley breeding, or landraces could be used directly for commercial barley production.

Leaf stripe, caused by the fungus Pyrenophora graminea (Pg), is an important seed-borne disease of barley causing significant yield and quality losses worldwide. In this work, expression of phenylalanine ammonia-lyase (PAL) was monitored in infected leaves of two barley genotypes, Banteng (resistant) and Furat1 (susceptible) across four-time points of Pg systemic movement using qRT-PCR approach. Data showed that the systemic movement of the fungus was slower in the resistant cultivar than in the susceptible one, as confirmed by both microscopic examination and culturing on PDA media. qRT-PCR revealed that the expression patterns of PAL gene significantly increased in infected plants in comparison with non-infected controls. Its expression correlated with Pg systemic movement in the root, stem and leaf fractions of the resistant and susceptible barley cultivars. Taken together, based on the Pg systemic movement within barley plants with different resistance levels, our data strengthen the idea that PAL plays a role in barley leaf stripe reduction. [ABSTRACT FROM AUTHOR]

This study aims at monitoring the dynamics of the occurrence and evolution of the attack of some pathogenic agents to barley, among which we mention: mildew (Blummeria graminis f.sp. hordei), leaf stripe (Pyrenophora graminea) and barley's rust (Puccinia hordei). Also, the influence of applying these fungicides on the harvest was monitored, as compared to the untreated control variant. For this study, an experiment with 6 treatment variants was created, in which the following phytosanitary products were used, as follows: ACANTO PLUS (picoxystrobin 200 g/l + cyproconazole 80 g/l); MYSTIC 250 EC (tebuconazole 250 g/l); FALCON PRO (prothioconazole 53 g/l+tebuconazole 148 g/l + spiroxamine 224 g/l); CAPALO (fenpropimorph 200g/l, epoxiconazole 62.5 g/l, metrafenon 75g/l). The treatment variants were the following: V1 - ACANTO PLUS 0.5 L/hA, 1 treatment applied at spike's release - blooming (5.05. 2019); V2 - ACANTO PLUS 0.5 L/HA, 1 treatment applied in "bellows" phase (20.04.2019) + 1 treatment applied at the beginning of kernel's filling (27.05.2019); V3 - CAPALO 1.0 L/HA, 1 treatment applied at spike's release -blooming (5.05. 2019); V4 - MYSTIC 250 EC 0.5 L/HA + 1 CAPALO 0.5 L/HA treatment applied in "bellows" phase (20.04.2019) + 1 treatment applied at the beginning of kernel's filling (27.05.2019); V5 - FALCON PRO 0.6 L/HA, 1 treatment applied at spike's release - blooming (5.05.2019); V6 - ACANTO PLUS 0.5 L/HA +- FALCON PRO 0.5 L/HA, 1 treatment applied in "bellows" phase (20.04.2019), 1 treatment applied at the beginning of kernel's filling (27.05. 2019), V7 - Untreated control variant. The experiment was placed in Latin square; the 7 variants being placed in 7 repetitions. The first two leaves placed under the spike had been analysed for the above. Among the pathogenic agents under monitoring, the greatest attacks were produced by Pyrenophora graminea fungus which produces barley leaf stripe disease. The harvests of the variants were the following: V1 - 5634 kg/ha, V2 - 5951 kg/ha, V3 - 5669 kg/ha, V4 -5658 kg/ha, V5 - 5494kg/ha, V6 - 5704 kg/ha and V7 (untreated control variant) - 5506 kg/ha. [ABSTRACT FROM AUTHOR]

Net blotch and leaf stripe caused by Pyrenophora teres and Pyrenophora graminea, respectively, are economical diseases of barley worldwide. Relatively little is known about the defense responses of barley challenged with these two closely related species. In the current work, expression of two well known defense-related genes PAL and PR2 were monitored in resistant and susceptible barley cultivars at early points of infection using quantitative real-time PCR (qPCR). Data showed significant variance in the expression patterns of the both genes between barley P. teres or P. graminea interactions as compared to the non-inoculated controls. It is also noteworthy that PAL and PR2 genes have a higher constitutive expression and faster induction in the resistant cultivar as compared with the susceptible one after infection with each pathogen. However, PCR (qPCR) analysis revealed higher gene expression in resistant barley plants inoculated with P. graminea as compared with P. teres, with a maximum expression for PR2 (13.8 and 6.41-fold) and PAL (14.8 and 7.89-fold) respectively, at 6 days post inoculation. The obtained results suggest that PAL and PR2 genes, positively regulate P. teres and P. graminea—resistance in barley plants during disease progress, which can provide testable hypotheses that will need direct future tests to determine how these changes may be specified in the genome of these closely related Pyrenophora species. [ABSTRACT FROM AUTHOR]

Pyrenophora graminea is the main fungal species associated with barley leaf stripe disease worldwide. Even though a heterothallic mating strategy has been proven for P. graminea, this species is mainly known based on the asexual morph in nature and there is no available information on the prevalence of an active sexual cycle within the populations of this species in Iran as well as many other barley-producing countries. The feasibility of a cryptic sexual cycle within Iranian isolates of P. graminea was assessed through analyzing the distribution and frequency of the mating type alleles on micro-spatial and macro-geographical scales. A total number of 306 P. graminea isolates were obtained from 93 fields in 45 geographical regions across East Azerbaijan province during 2016–2017. A multiplex PCR assay was developed for simultaneous identification of P. graminea and screening of its mating type alleles using previously designed primer sets. Using the multiplex PCR assay, a 435-bp band was consistently amplified from all P. graminea isolates; while a 1300-bp fragment or a 1150-bp fragment was only amplified from the isolates harboring MAT-1 and MAT-2 alleles, respectively. The mating type identity of 164 isolates was determined as MAT-1 and 142 isolates as MAT-2. Results of the present study revealed a nearly equal distribution (1:1 ratio; X2=1.582) of mating type alleles within and between different populations of P. graminea. Results of the micro-spatial and macro-geographical distribution of mating types showed that both mating types were often present on almost all studied scales, including: within the same lesion of each leaf from a single barley plant, from the same field or different fields in the same region, and from different regions. Based on the results of the current study and referring to the earlier reports on the population structure of P. graminea, it is concluded that this pathogen undergoes regular cycles of sexual recombination in most of the examined regions. [ABSTRACT FROM AUTHOR]

Wild barley (Hordeum spontaneum) and barley landraces are important sources of genetic variation for disease resistance. Thirty wild barley (H. spontaneum) genotypes and 30 barley landraces were evaluated for susceptibility to two Drechslera graminea isolates. Virulence differences were observed between the isolates, while the responses of the host genotypes to the isolates also varied. Of the H. spontaneum genotypes, 23% and 63%, respectively, were resistant to the Yozgat D. graminea isolate, and Eskişehir D. graminea isolates. On the other hand, 43% and 90% of the barley landraces were resistant to Yozgat and Eskişehir D. graminea isolates, respectively. Hordeum spontaneum genotypes 13, 24, 27, 29, 54, 86, and 91 exhibited resistance to both D. graminea isolates, while genotypes 14 and 32 showed intermediate reactions to the Yozgat isolate and resistant reactions to the Eskişehir isolate. Barley landraces 21, 37, 38, 39, 40, 73, 98, 128, 139, 153, 159,167, and 171 showed resistant reactions, and barley landrace 8 showed an intermediate reaction to both isolates. Barley landraces 3, 20, 24, 71, 101, 103, 104 and 160 exhibited intermediate responses to the Yozgat isolate and a resistant response to the Eskişehir isolate. Using resistant barley genotypes would reduce the need for pesticides for control of leaf stripe, and be an environmentally preferred strategy for disease control. The disease resistance present in wild barley and barley landraces are important for expanding the genetic basis of cultivated barley (H. vulgare). The resistant and intermediate genotypes identified in this study could be used as resistance sources in barley breeding, or landraces could be used directly for commercial barley production. [ABSTRACT FROM AUTHOR]

The seed-borne (Pyrenophora graminea; Pg) and foliar (Blumeria graminis; Bg) are two economically important fungal pathogens of barley worldwide. Barley plant resistance genes, as the pathogenesis related proteins play an important role in defense mechanisms. This study aimed to monitor the expression of PR2 and PAL pathogenesis related genes during compatible/incompatible barley interaction with Pg and Bg at different time points of disease development using the Quantitative Real-time PCR technique (qRT-PCR). Comparison of data showed that PR2 and PAL were significantly over expressed in infected resistant and susceptible plants as against their lower expression in controls,. Upregulation of these defense-related genes during Pg and Bg infections was companied with a slow development of disease symptoms at the time course in the resistant genotype. qRT-PCR analysis revealed higher gene expression in resistant barley plants inoculated with Pg as compared with Bg, with a maximum expression for PR2 (13.8 and 5.06-fold) and PAL (14.8 and 4.51-fold) respectively, at the latest stage of each disease development. It was also noteworthy that PR2 and PAL genes, had higher constitutive expression and faster induction for the both pathogens in the resistant genotype as compared with the susceptible one. Obtained results suggest that both genes, PR2 and PAL, positively regulate Pg- and Bg-resistance in barley plants during disease progress. These expression patterns can provide useful insights to better understanding of the barley–fungus interactions with different fungal lifestyles. [ABSTRACT FROM AUTHOR]

Barley leaf stripe, caused by the fungal pathogen Pyrenophora graminea, is becoming a destructive disease in the Hexi corridor of the Gansu province (China). Knowledge of the genetic diversity, population structure, and specific pathogenicity of P. graminea is essential for barley resistance breeding and efficient control of leaf stripe. In this study, whole-genome sequences of P. graminea were applied to identify microsatellites and develop simple sequence repeat (SSR) markers. These SSR markers were used to examine the genetic diversity and population structure of barley leaf stripe fungus in China. A total of 403 SSR primers were designed and synthesized, and target fragments were amplified by 390 microsatellite markers. These fragments corresponded to 789 alleles (1–5 alleles per locus) in eight strains (average, 2.02 alleles per marker locus). The cluster analysis and population structure were analyzed by means of 100 markers, and a total of 696 alleles were detected from 44 accessions (mean, 6.96 alleles per marker; range, 3–17). The average gene diversity was 0.63, and ranged from 0.11 to 0.92. The polymorphism information content (PIC) ranged from 0.11 to 0.91 with an average of 0.59. The Shannon's Information Index ranged from 0.29 (for Pgm254 and Pgm337) to 2.64 (for Pgm371), with an average of 1.36. Results of cluster analysis showed that 44 accessions were divided into four clusters. Overall, 44 strains were divided into three subpopulations, comprising 19, 17, and eight strains each. Six isolates were tested for pathogenicity differentiation on 10 barley cultivars. Cultivar Ganpi2 was found to be resistant to all six isolates; nine of 10 cultivars had varied reactions to six isolates. Genome sequencing of P. graminea enabled SSR marker development, and SSR markers are applicable to studies of population genetic structure in P. graminea. We conclude that pathogenicity differentiation exists among the six isolates in the study. [ABSTRACT FROM AUTHOR]

Abstract The high-osmolarity glycerol (HOG) signaling pathway regulates the adaptation of fungi to environmental stressors. The mitogen-activated protein kinase kinase (MAPKK) PBS2 of Saccharomyces cerevisiae serves as a scaffold protein in the HOG pathway. We characterized the pgpbs gene of Pyrenophora graminea , which encodes a MAPKK that is 56% orthologous to PBS2 of S. cerevisiae. A cloning technique based on homology was applied to amplify the pgpbs gene. Specific silent mutations then were generated in pgpbs. We evaluated the potential roles of PGPBS in the osmotic response, vegetative differentiation, cell wall integrity, drug resistance, and pathogenicity. Our findings indicated that the pgpbs coding region comprises 2075 base pairs and encodes a protein of 676 amino acids. Mutants deficient in pgpbs expression had significant reductions in vegetative growth and were sensitive to calcofluor white (CFW), an inhibitor of cell wall synthesis. Mutants also lost pathogenicity and were sensitive to an osmotic stress–inducing medium containing NaCl and sorbitol. Moreover, mutants had increased resistance to the dicarboximide fungicide iprodione and the triazole fungicide tebuconazole. These findings suggest that pgpbs is involved in the osmotic and ionic stress responses, vegetative differentiation, cell wall integrity, virulence, and tolerance to iprodione and tebuconazole. We expect that our findings will help elucidate the pathogenesis of barley leaf stripe and will inform strategies for breeding resistance to this disease. Highlights • The pgpbs gene was first element of the TCS pathway been characterized in Pyrenophora graminea. • An pgpbs gene silent mutations of a Pyrenophora graminea was first constructed by RNA interference. • The pgpbs gene is involved in the osmotic and ionic stress responses, vegetative differentiation, cell wall integrity. • The pgpbs gene is involved in virulence and tolerance to iprodione and tebuconazole. • The pgpbs gene played a key role in pathogenesis of Pyrenophora graminea. [ABSTRACT FROM AUTHOR]

Wheat grains are inhabited by different fungi, including plant pathogens and fungi - mycotoxin producers. The composition of seed mycobiota can be influenced by different factors, including agronomic practices, but the results are still contradictory. The aim of this study was to evaluate the mycobiota of wheat grains depending on agroecological conditions. Wheat grains were obtained from a two-factorial field trial: A - tillage system (A1 - ploughing at a depth of 22-24 cm; A2 - harrowing at a depth of up to 10 cm); B - crop rotation (B1 - continuous wheat; B2 - oilseed rape and wheat; B3 - crop rotation). The mycobiota of grain were determined by mycological and molecular methods. The most abundant and widespread of the mycobiota were Pyrenophora tritici-repentis, Alternaria spp., Arthrinium spp., and Fusarium avenaceum. Higher amounts of precipitation increased the infection of grains with Fusarium fungi. Seven species of Fusarium were identified in the grain samples: F. avenaceum, F. poae, F. graminearum, F. culmorum, F. acuminatum, F. sporotrichioides, and F. tricinctum. The soil tillage method and crop rotation did not influence the total incidence of Fusarium spp., but the abundance of a particular species differed depending on agronomic practice. The research suggests that continuous wheat sowing under conditions of reduced soil tillage can increase the level of risk of grain infection with F. graminearum and, consequently, the accumulation of mycotoxins. [ABSTRACT FROM AUTHOR]

The usefulness of IRAP (inter-retrotransposon amplified polymorphism) and ITS-RFLP (restriction of PCR-amplified internal transcribed spacers of the rDNA) markers in the analysis of 39 Pyrenophora graminea isolates was determined. Each marker system could discriminate between all of the isolates in detecting polymorphism, albeit with variable efficiency. IRAP and ITS-RFLP produced 85% and 77% polymorphic bands, respectively, with a corresponding mean polymorphic information content (PIC) of 0.38 and 0.36. The IRAP marker index ratio (2.41) was higher than ITS-RFLP (1.50). On one hand, the quality nature of data (QND) was higher for ITS-RFLP (0.169) than IRAP (0.093). However, correlation between both marker similarity matrices was significant (r = 0.34, p < 0.05). These findings suggest their combined use in phylogenetic analysis. To our knowledge, this is the first report of a comparison involving these two advanced DNA marker systems.

Seedling reactions of 15 barley cultivars grown in Turkey were determined under greenhouse conditions to five isolates of Drechslera graminea, the causal agent of barley stripe disease. Isolates were collected from different parts of Ankara province. Differences were observed among the reactions of the cultivars to the isolates of the fungus. There were also pathogenicity differences among the isolates. The cultivars Çumra 2001 and Yerçil 147 were resistant to all five isolates. Cultivar Sladoran was resistant to 4 isolates. The cultivars Erginel 90, Orza 96, Çetin 2000 and Aydanhanım were susceptible to three isolates of the fungus. The reactions of other varieties ranged between resistant and susceptible depending on the isolates. Isolate Dg3 was the most virulent

The barley is under attacked of many pathogenic agents during the vegetation period. Out of these pathogenic agents, the Pyrenophora graminea fungus had been producing barley's leaf stripe disease in the Eastern Baragan area, in 2016 (Velichi E. 2012). An experiment was created in the year 2016, using 4 products of phytosanitary usage, as follows: ACANTO PLUS (picoxistrobin + cyproconazole), MYSTIC 250 EC (tebuconazole); BUMPER 250 EC (propiconazole) and TOPSIN 500 SC (tiophanate-methyl) (Pest - Expert). This experiment consisted in 7 variants (6 variants with phytosanitary treatments in different combinations of products, plus a control sample not treated). The experiment was placed in randomised blocks. The 7 variants were placed in 6 repetitions. Out of the pathogenic agents monitored, the biggest attacks had been produced by Pyrenphora graminea fungus which produces leaf stripe at barley. For this, there had been analysed the first two leaves under the spike. The observations had shown that for all 6 variants of treatment, the attack degree (GA %) of the disease (barley's leaf stripe) was more reduced than at the control sample not treated. The productions of the variants treated (V1 ... V6) had been higher than the production of the control sample not treated, V7. The productions of some variants of treatment had been significantly higher than the production of the control sample not treated (statistic insurance DL 5%). In what concerns the economic efficiency, the fungicide product MYSTIC 250 EC 0.5l/ha had proven to be the most efficient in what price was concerned. [ABSTRACT FROM AUTHOR]

Background: Barley leaf stripe disease, caused by the fungus Pyrenophora graminea (Pg), is a worldwide crop disease that results in significant loss of barley yield. The purpose of the present work was to use transcriptomic profiling to highlight barley genes and metabolic pathways affected or altered in response to Pg infection and consequently elucidate their involvement and contribution in resistance to leaf stripe. Results: Our study examined and compared the transcriptomes of two barley genotypes using an established differential display reverse-transcription polymerase chain reaction (DDRT-PCR) strategy at 14 and 20 days post-inoculation (dpi). A total of 54 significantly modulated expressed sequence tags (ESTs) were identified. The analysis of gene expression changes during the course of infection with Pg suggested the involvement of 15 upregulated genes during the immunity response. By using network-based analyses, we could establish a significant correlation between genes expressed in response to Pg invasion. Microscopic analysis and quantitative PCR (qPCR) profiling of callose synthase and cellulose synthases revealed a direct involvement of cell wall reinforcement and callose deposition in the Pg-resistant phenotype. Conclusions: We have identified a number of candidate genes possibly involved in the host-pathogen interactions between barley and Pg fungus, 15 of which are specifically expressed in Pg-resistant plants. Collectively, our results suggest that the resistance to leaf stripe in barley proceeds through callose deposition and different oxidation processes. [ABSTRACT FROM AUTHOR]

A total of 19 Iranian native barley cultivars were in artificial infection experiments tested for resistance against isolate19 of the leaf stripe fungs, Pyrenophora graminea. The cultivar Eram was immune against isolate 19 and the cultivarsJonob, Makoy, C2, showed high resistance with less than 6% plant infected, the cultivars Zarjo, Valfajr, Reyhan, Karun,Karun × Kavir were the most susceptible cultivars.

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool. [ABSTRACT FROM AUTHOR]

The study reported here primarily focuses on whether fruit extracts of Azadirachta indica Juss. (Neem) can induce systemic acquired resistance (SAR) in Hordeum vulgare against Drechslera graminea. A single leaf from each of the 1-month-old seedlings grown in 50 pots was treated with neem extract. Seven samples were collected at 12-h intervals for estimation of salicylic acid (SA) content and activities of phenylalanine ammonia lyase (PAL) and peroxidase (PO). Disease incidence was recorded on uninoculated leaves after 2 weeks and on newly emerged leaves after 3 weeks of inoculation of spores of the pathogen. Treatment of single leaf of barley seedlings with aqueous fruit extract of neem could protect the untreated and later emerging leaves of these seedlings from infection by leaf stripe pathogen. The concentration of SA and activities of PAL and PO were significantly higher in untreated leaves of seedlings given a single leaf treatment with neem fruit extract. The results show that neem fruit extract induced SAR in barley seedlings against D. graminea. The results of the study are significant for developing an environment-friendly biocide, which could induce SAR in crop plants leading to efficient management of pathogens [ABSTRACT FROM AUTHOR]

Various 2-N-acyl-5-methylisoxazolone derivatives were prepared, and their antifungal activities were evaluated in vitro with mycelial growth inhibition tests. In contrast with N-alkyl derivatives, the acyl compounds showed significant activity against Pyrenophora graminea, Fusarium graminearum, Alternaria alternata, Cercospora beticola, Rhynchosporium secalis, Septoria tritici, Microdochium nivale, Rhizoctonia solani and Gaeumannomyces graminis. Of note, cinnamoyl, 3-furan-3-ylacryloyland 3-thiophen-3-yl-acryloylamides, and t-butylacetyl and pivaloyl derivatives showed high inhibition rates at 25 mg/L against R. solani and G. graminis, respectively. [ABSTRACT FROM AUTHOR]

Xylanase and amylase have gained increasing interest because of their various biotechnology applications. In this research, the restriction of PCR-amplified internal transcribed spacers (ITS) of ribosomal DNA (rDNA) was used to confirm the genetic variation among 22 isolates of Pyrenophora graminea differing in their xylanase and amylase production. The fingerprints generated from the six restriction digestions of the rDNA ITS region showed high levels of intraspecific variation within the P. graminea population. Neighbour-Joining diagram, based on Nei's genetic distances, showed that isolates formed two phylogenetic groups. No apparent association could be observed between xylanase and amylase production and genetic diversity among the twenty-two isolates. [ABSTRACT FROM AUTHOR]

The inter-retrotransposon amplified polymorphism (IRAP) was used to confirm the genetic variation among 22 strains of Pyrenophora graminea differing in their xylanase production. A total of 162 bands were scored of which 151 (93.21%) were polymorphic. The molecular parameter used showed that P. graminea strains reside in four phylogenetic groups. There was observed the resolution between clustering strains and their xylanase production. Hence, the described approach presented here constitutes no prior assumption about the characterization of P. graminea strains differing in xylanase production. [ABSTRACT FROM AUTHOR]

Background: Leaf stripe disease on barley (Hordeum vulgare) is caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea. Race-specific resistance to leaf stripe is controlled by two known Rdg (Resistance to Drechslera graminea) genes: the H. spontaneum-derived Rdg1a and Rdg2a, identified in H. vulgare. The aim of the present work was to isolate the Rdg2a leaf stripe resistance gene, to characterize the Rdg2a locus organization and evolution and to elucidate the histological bases of Rdg2a-based leaf stripe resistance. Principal Findings: We describe here the positional cloning and functional characterization of the leaf stripe resistance gene Rdg2a. At the Rdg2a locus, three sequence-related coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NB-LRR) encoding genes were identified. Sequence comparisons suggested that paralogs of this resistance locus evolved through recent gene duplication, and were subjected to frequent sequence exchange. Transformation of the leaf stripe susceptible cv. Golden Promise with two Rdg2a-candidates under the control of their native 5' regulatory sequences identified a member of the CC-NB-LRR gene family that conferred resistance against the Dg2 leaf stripe isolate, against which the Rdg2a-gene is effective. Histological analysis demonstrated that Rdg2a-mediated leaf stripe resistance involves autofluorescing cells and prevents pathogen colonization in the embryos without any detectable hypersensitive cell death response, supporting a cell wall reinforcement-based resistance mechanism. Conclusions: This work reports about the cloning of a resistance gene effective against a seed borne disease. We observed that Rdg2a was subjected to diversifying selection which is consistent with a model in which the R gene co-evolves with a pathogen effector(s) gene. We propose that inducible responses giving rise to physical and chemical barriers to infection in the cell walls and intercellular spaces of the barley embryo tissues represent mechanisms by which the CC-NB-LRR-encoding Rdg2a gene mediates resistance to leaf stripe in the absence of hypersensitive cell death. [ABSTRACT FROM AUTHOR]

Leaf stripe of barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts. Two barley segregating populations represented by 103 recombinant inbred lines (RILs) of the cross L94 (susceptible) × Vada (resistant) and 194 RILs of the cross Arta (susceptible) × Hordeum spontaneum 41-1 (resistant) were analysed with two highly virulent leaf stripe isolates, Dg2 and Dg5, to identify loci for P. graminea resistance. A major gene with its positive allele contributed by Vada and H. spontaneum 41-1 was detected in both populations and for both pathogen isolates on chromosome 2HL explaining 44.1 and 91.8% R2, respectively for Dg2 and Dg5 in L94 × Vada and 97.8 and 96.1% R2, respectively for Dg2 and Dg5 in Arta × H. spontaneum 41-1. Common markers in the gene region of the two populations enabled map comparison and highlighted an overlapping for the region of the resistance locus. Since the map position of the resistance locus identified in this report is the same as that for the leaf stripe resistance gene Rdg1a, mapped earlier in Alf and derived from the ‘botanical’ barley line H. laevigatum, we propose that leaf stripe resistance in Vada and H. spontaneum 41-1 is governed by the same gene, namely by Rdg1a, and that Rdg1a resistance could be traced back to H. spontaneum, the progenitor of cultivated barley. PCR-based molecular markers that can be used for marker-assisted selection (MAS) of Rdg1a were identified. An Rdg1a syntenic interval with the rice chromosome arm 4L was identified on the basis of rice orthologs of EST-based barley markers. Analysis of the rice genes annotated into the syntenic interval did not reveal sequences strictly belonging to the major class (nucleotide-binding site plus leucine-rich repeat) of the resistance genes. Nonetheless, four genes coding for domains that are present in the major disease-resistance genes, namely receptor-like protein kinase and ATP/GTP-binding proteins, were identified together with a homolog of the barley powdery mildew resistance gene mlo. Three (out of five) homologs of these genes were mapped in the Rdg1a region in barley and the mlo homolog map position was tightly associated with the LOD score peak in both populations. [ABSTRACT FROM AUTHOR]

Barley stripe disease caused by Drechslera graminea is an important disease of barley worldwide. During 2012, 205 barley fields were surveyed for the presence of this disease in Central Anatolia, Turkey. Eighty-two fields (40%) were found to be infected with Drechslera graminea. Disease incidence ranged between 1-70%. Generally low amount of disease was observed, however, disease incidence was high (up to 70%) in some fields of Kayseri, Sivas, Yozgat and Aksaray provinces. Large differences were observed among provinces for the proportion of crops infected. [ABSTRACT FROM AUTHOR]

Barley ( Hordeum vulgare L.) leaf stripe is caused by the seed-borne fungus Pyrenophora graminea. We investigated microscopically and molecularly the reaction of barley embryos to leaf stripe inoculation. In the resistant genotype NIL3876- Rdg2a, fungal growth ceased at the scutellar node of the embryo, while in the susceptible near-isogenic line (NIL) Mirco- rdg2a fungal growth continued past the scutellar node and into the embryo. Pathogen-challenged embryos of resistant and susceptible NILs showed different levels of UV autofluorescence and toluidine blue staining, indicating differential accumulation of phenolic compounds. Suppression subtractive hybridization and cDNA amplified fragment-length polymorphism (AFLP) analyses of embryos identified P. graminea-induced and P. graminea-repressed barley genes. In addition, cDNA-AFLP analysis identified six pathogenicity-associated fungal genes expressed during barley infection but at low to undetectable levels during growth on artificial media. Microarrays representing the entire set of differentially expressed cDNA-AFLP fragments and 100 barley homologues of previously described defence-related genes were used to study gene expression changes at 7 and 14 days after inoculation in the resistant and susceptible NILs. A total of 171 significantly modulated barley genes were identified and assigned to four groups based on timing and genotype dependence of expression. Analysis of the changes in gene expression during the barley resistance response to leaf stripe suggests that the Rdg2a-mediated response includes cell-wall reinforcement, signal transduction, generation of reactive oxygen species, cell protection, jasmonate signalling and expression of plant effector genes. The identification of genes showing leaf stripe inoculation or resistance-dependent expression sets the stage for further dissection of the resistance response of barley embryo cells to leaf stripe. [ABSTRACT FROM AUTHOR]

The main aim of this study was to test the patterns of sequence divergence and haplotype structure at the MAT locus of Pyrenophora teres, the causal agent of barley ‘net blotch’ disease. P. teres is a heterothallic ascomycete that co-occurs in two symptomatological forms, the net form (NF) and the spot form (SF). The mating-type genes MAT1-1-1 and MAT1-2-1 were sequenced from 22 NF isolates (12 MAT1-1-1 and 10 MAT1-2-1 sequences) and 17 SF isolates ( 10 MAT1-1-1 and seven MAT1-2-1 sequences) collected from Sardinian barley landrace populations and worldwide. On the basis of a parsimony network analysis, the two forms of P. teres are phylogenetically separated. More than 85% of the total nucleotide variation was found between formae speciales. The two forms do not share any polymorphisms. Six diagnostic nucleotide polymorphisms were found in the MAT1-1-1 intron (1) and in the MAT1-1-1 (3) and MAT1-2-1 (2) exons. Three diagnostic non-synonymous mutations were found, one in MAT1-1-1 and two in MAT1-2-1. For comparison with P. teres sequence data, the mating-type genes from Pyrenophora graminea were also isolated and sequenced. Divergence between P. graminea and P. teres is of a similar magnitude to that between NF and SF of P. teres. The MAT genes of P. graminea were closer to those of SF than to NF, with the MAT1-2-1 SF peptide not different from the MAT1-2-1 peptide of P. graminea. Overall, these data suggest long genetic isolation between the two forms of P. teres and that hybridization is rare or absent under field conditions, with each form having some particular niche specialization. This indicates that research on resistance to P. teres should consider the two forms separately, as different species. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to examine the effect of Melaleuca alternifolia essential oil (TTO) and its principal components on four cereal-pathogenic fungi. Methods and Results: The antimycotic properties of TTO and of terpinen-4-ol, γ-terpinen and 1,8-cineole (eucalyptol) were evaluated in vitro on Fusarium graminearum, Fusarium culmorum and Pyrenophora graminea. Moreover, barley leaves infected with Blumeria graminis were treated with whole TTO. All the tested fungi were susceptible to TTO and its components. Conclusions: TTO exerted a wide spectrum of antimycotic activity. Single TTO purified components were more active than the whole oil in reducing in vitro growth of fungal mycelium and, among the tested compounds, terpinen-4-ol was the most effective. Significance and Impact of the Study: TTO and its components can be considered potential alternative natural fungicides. [ABSTRACT FROM AUTHOR]

The naked/hulled kernel trait is controlled in barley by a single gene called nud, on chromosome 7H. The first aim of this work was use bulked segregant analysis to find, new PCR-based markers linked to nud for marker-assisted selection (MAS). A new SCAR marker (sJ14) was developed, which is useful for introgressing the naked trait. This, and three other SCARs, were placed on the ‘Proctor’ × ‘Nudinka’ map to detail a 0.9-cM fragment tagging nud. In order to evaluate the haplotypes around the nud locus, a phenotypically differentiated collection of naked/hulled genotypes was characterized by means of the above markers. Eight different marker haplotypes were found in the breeding germplasm, and a new allele for the marker sKT7 was found. The same barley collection has been surveyed for resistance/susceptibility to leaf stripe ( Pyrenophora graminea), in order to investigate any possible association between this and other traits. The naked/hulled seed trait was not associated with resistance/susceptibility to the fungus. [ABSTRACT FROM AUTHOR]

Barley-pathogenic Pyrenophora isolates are P. graminea (PG), P. teres f. maculata (PTM) and P. teres f. teres (PTT), which cause foliar leaf stripe, spot blotch and net blotch lesions, respectively. However, the species are often indistinguishable by morphological and cultural characteristics. Random amplified polymorphic DNA (RAPD) analysis has been used to study the genetic relationship amongst 11 PG, 9 PTM and 23 PTT isolates from distant geographical locations. Using seven primers, 55 (52.38%) polymorphic DNA bands were detected out of 105 different fragments amplified in the three pathogens. Genotypic diversity was high as all but two PTT strains had distinct multilocus RAPD fingerprints. Unweighted pair-group method with arithmetic average (UPGMA) clustering separated the isolates into three main clusters, corresponding to the three pathogens studied. No clear geographical substructuring was found. Nei's gene diversity analysis detected only small differences (max. 6.6%) in band frequencies but considerable levels of differentiation were observed among the pathogen species/forms. However, the variability among the Pyrenophora species/forms (max. 42.0%) was less than within species/forms (max. 58%). Nei's unbiased genetic distance values were in agreement with UPGMA clustering and gene diversity analysis: the two forms of P. teres showed higher divergence from one another ( D = 0.132) than the distance found between PG and PTM ( D = 0.094). The results suggest that the present taxonomical classification of these morphological taxa may not correspond to their phylogenetic relationship and that there is a very close genetic relationship amongst barley-pathogenic Pyrenophora species, but genetic exchanges between them could be infrequent. [ABSTRACT FROM AUTHOR]