<i>Pyrenophora teres</i> Drechs. (anamorph: <i>Drechslera teres</i> (Sacc.) Shoem.) is the causal agent of barley net blotch. Net blotch is an economically important disease commonly found throughout the barley producing regions of the world. Significant financial losses result from yield reductions, ranging from 15-35%, and decreased grain quality. Despite its prevalence, it is unclear if the P. teres-barley pathosystem follows a gene-for-gene model, and more generally, little is known about its genetic organization. Three studies were initiated to address these questions.<p>The first study investigated the genetic control of avirulence in <i>P. teres.</i> To establish an appropriate study system, a collection of ten net form (<i>P. teres f. teres</i>) and spot form (<i>P. teres f. maculata</i>) isolates were evaluated on a set of eight differential barley lines to identify two isolates with differential virulence on a specific host line. WRS 1906, exhibiting low virulence on the cultivar Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for virulence on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (Chi-square = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism (AFLP) markers closely linked to the avirulence gene (AvrHeartland). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model.<p>In the second study, five isolates of P. teres, representing both net and spot forms, were analyzed by the germ tube burst method (GTBM) and pulsed field gel electrophoresis (PFGE) to determine the species karyotype. Nine chromosomes were observed in all isolates using the GTBM and estimation of chromosome lengths varied from 0.5 to 3.0 µm. PFGE separated 7 to 8 bands depending on isolate, but analysis of bands by densitometry indicated nine chromosomes. Chromosome size ranged from 1.8 to ~6.0 Mb providing a genome size estimate of 32 to 39 Mb. Significant chromosome-length polymorphisms (CLP) were observed between isolates. These CLP did not hinder mating between mating-type compatible net form isolates. No particular CLP or individual chromosome could be associated with differences in disease symptoms observed between pathogen forms. This study provides the first karyotype of both P. teres forms and will assist genetic mapping of this pathogen.<p>A genetic linkage map of <i>P. teres f. teres</i>, was constructed in the third study using the population of 67 progeny derived from the WRS 1906  WRS 1607 cross. The map consists of 138 markers including 114 AFLPs, 21 telomere RFLPs, the mating-type (MAT) locus and an avirulence locus (AvrHeartland) controlling interaction with barley cultivar Heartland. Markers were distributed across 24 linkage groups ranging in length from 2 to 110 cM with an average marker interval of 8.5 cM. The total map length was 797 cM. A telomere-specific probe, consisting of the sequence (TTAGGC)4, was used to map 15 of the 18 telomeres. One of these telomeres mapped to within 3 cM of the AvrHeartland locus. Attempts to consolidate linkage groups by hybridizing markers to the electrophoretically separated chromosomes was unsuccessful because probes bound to multiple chromosomes, likely due to repetitive DNA within the probe. This is the first genetic map reported for this species and it will be a useful genetic tool for map-based cloning of the AvrHeartland gene tagged in this study. <p>This research has provided a number of new insights into the net blotch pathogen and provides a useful research tool in the form of a genetic map. This information lays the foundation for further genetic study of P. teres and will complement studies on barley resistance to net blotch that may potentially lead to more durable resistance.

<i>Pyrenophora teres</i> Drechs. (anamorph: <i>Drechslera teres</i> (Sacc.) Shoem.) is the causal agent of barley net blotch. Net blotch is an economically important disease commonly found throughout the barley producing regions of the world. Significant financial losses result from yield reductions, ranging from 15-35%, and decreased grain quality. Despite its prevalence, it is unclear if the P. teres-barley pathosystem follows a gene-for-gene model, and more generally, little is known about its genetic organization. Three studies were initiated to address these questions.<p>The first study investigated the genetic control of avirulence in <i>P. teres.</i> To establish an appropriate study system, a collection of ten net form (<i>P. teres f. teres</i>) and spot form (<i>P. teres f. maculata</i>) isolates were evaluated on a set of eight differential barley lines to identify two isolates with differential virulence on a specific host line. WRS 1906, exhibiting low virulence on the cultivar Heartland, and WRS 1607, exhibiting high virulence, were mated and 67 progeny were isolated and phenotyped for virulence on Heartland. The population segregated in a 1:1 ratio, 34 avirulent to 33 virulent (Chi-square = 0.0, P = 1.0), indicating single gene control of WRS 1906 avirulence on Heartland. Bulked segregant analysis was used to identify six amplified fragment length polymorphism (AFLP) markers closely linked to the avirulence gene (AvrHeartland). This work provides evidence that the P. teres-barley pathosystem conforms to the gene-for-gene model.<p>In the second study, five isolates of P. teres, representing both net and spot forms, were analyzed by the germ tube burst method (GTBM) and pulsed field gel electrophoresis (PFGE) to determine the species karyotype. Nine chromosomes were observed in all isolates using the GTBM and estimation of chromosome lengths varied from 0.5 to 3.0 µm. PFGE separated 7 to 8 bands depending on isolate, but analysis of bands by densitometry indicated nine chromosomes. Chromosome size ranged from 1.8 to ~6.0 Mb providing a genome size estimate of 32 to 39 Mb. Significant chromosome-length polymorphisms (CLP) were observed between isolates. These CLP did not hinder mating between mating-type compatible net form isolates. No particular CLP or individual chromosome could be associated with differences in disease symptoms observed between pathogen forms. This study provides the first karyotype of both P. teres forms and will assist genetic mapping of this pathogen.<p>A genetic linkage map of <i>P. teres f. teres</i>, was constructed in the third study using the population of 67 progeny derived from the WRS 1906  WRS 1607 cross. The map consists of 138 markers including 114 AFLPs, 21 telomere RFLPs, the mating-type (MAT) locus and an avirulence locus (AvrHeartland) controlling interaction with barley cultivar Heartland. Markers were distributed across 24 linkage groups ranging in length from 2 to 110 cM with an average marker interval of 8.5 cM. The total map length was 797 cM. A telomere-specific probe, consisting of the sequence (TTAGGC)4, was used to map 15 of the 18 telomeres. One of these telomeres mapped to within 3 cM of the AvrHeartland locus. Attempts to consolidate linkage groups by hybridizing markers to the electrophoretically separated chromosomes was unsuccessful because probes bound to multiple chromosomes, likely due to repetitive DNA within the probe. This is the first genetic map reported for this species and it will be a useful genetic tool for map-based cloning of the AvrHeartland gene tagged in this study. <p>This research has provided a number of new insights into the net blotch pathogen and provides a useful research tool in the form of a genetic map. This information lays the foundation for further genetic study of P. teres and will complement studies on barley resistance to net blotch that may potentially lead to more durable resistance.

Pyrenophora teres f. teres is the causal agent of net form net blotch of barley. P. teres f. teres is prevalent globally across all barley growing regions and globally is the most devastating foliar disease of barley. Though economically important, the molecular mechanism whereby P. teres f. teres causes disease is poorly understood and investigations into these mechanisms have been hindered by a lack of genomic resources. To set a genomic foundation for P. teres f. teres the reference isolate 0-1 was sequenced and assembled using PacBio single molecule real-time (SMRT) sequencing and scaffolded into 12 chromosomes to provide the first finished genome of P. teres f. teres. High confidence gene models were generated for the reference genome of isolate 0-1 using a combination of pure culture and in planta RNA sequencing. An additional four P. teres f. teres isolates were sequenced and assembled to the same quality as the reference isolate 0-1 and used in a comparative genomic study. Comparisons of the five P. teres f. teres isolates showed a two-speed genome architecture with the genome being partitioned into core and accessory genomic compartments. Accessory genomic compartments clustered in sub-telomeric regions of the P. teres f. teres genome with a majority of previously identified quantitative trait loci (QTL) associated with avirulence/virulence being spanned by these accessory regions. Using these genomic resources, with a bi-parental mapping population and a natural population for QTL analysis and genome wide association study (GWAS), respectively, we identified a candidate gene for the previously mapped AvrHar. QTL analysis identified a locus extending off the end of P. teres f. teres chromosome 5 and GWAS analysis identified significant associations with a gene encoding a small secreted protein. The candidate AvrHar gene was validated using CRISPR-Cas9-RNP gene disruption in parental isolates 15A and 0-1. Disruption of AvrHar in isolate 15A did not result in a phenotypic change while disruption of the 0-1 allele resulted in a complete loss of pathogenicity. This is the first identification of an effector from P. teres f. teres validated using CRISPR-Cas9-RNP gene editing. / North Dakota Barley Council

Net blotch is one of the most devastating diseases of barley (<i>Hordeum vulgare</i> L.) and occurs in two distinct forms, net-type net blotch (NTNB) and spot-type net blotch (STNB), caused by the fungal pathogens <i>Pyrenophora teres</i> f. sp. <i>teres</i> Smedeg. and <i>P</i>. <i>teres</i> f. sp. <i>maculata</i> Smedeg., respectively. Several sources of resistance have been previously reported, however, few barley cultivars with high levels of resistance have been developed from these sources. Efficient utilization of available resistance sources is dependent upon successful characterization of genes governing resistance in each resistant parent. Five net blotch resistant parents and one susceptible parent were crossed to identify novel resistance genes, postulate gene number and mode of inheritance, and conduct linkage mapping of novel genes for net blotch resistance. Results indicate that the highly resistant spring barley lines CIho 2291 and CIho 5098, and the winter barley cultivar Nomini each have single dominant genes for NTNB resistance. Resistance to NTNB in CIho 5098 is controlled by the same dominant gene conferring resistance in Nomini. Resistance to NTNB in CIho 2291 is controlled by one dominant gene which putatively is the same gene conferring resistance in ND B112, but differs from the resistance genes carried by the other parents in this study. An F2 population of 238 individuals derived from a cross between Nomini and the susceptible parent "Hector", and an F2 population of 193 individuals derived from a cross between CIho 2291 and Hector were used to map the genes governing NTNB resistance in Nomini and CIho 2291. The dominant gene governing resistance in Nomini, temporarily designated <i>Rpt-Nomini</i>, was mapped to a 9.2 cM region near the centromere of barley chromosome 6H between the flanking microsatellite markers Bmag0344a (r2=0.70) and Bmag0103a (r2=0.90), which were 6.8 cM and 2.4 cM away from <i>Rpt-Nomini</i>, respectively. The dominant gene governing resistance in CIho 2291, temporarily designated <i>Rpt-CIho2291</i>, was mapped to the distal region of barley chromosome 6H between the flanking microsatellite markers Bmag0173 (r2=0.65) and Bmag0500 (r2=0.26), which were 9.9 cM and 24.4 cM from <i>Rpt-CIho2291</i>, respectively. Previous studies have reported genes governing net blotch resistance in this region; however, allelism tests have not been conducted to determine the relationship between these genes. Identification of the chromosomal location of <i>Rpt-Nomini</i> and <i>Rpt-CIho2291</i> will facilitate future efforts in pyramiding multiple independent genes for net blotch resistance. / Ph. D.

The occurrence of spot type net blotch (Pyrenophora teres f. maculata) and net type net blotch (P. teres f. teres) as well as the occurrence of net blotch mating types has been investigated in Lithuania, Latvia and Estonia. Genetic diversity of barley net blotch isolates from various locations in Lithuania was investigated using ISSR and AFLP markers. The net blotch resistance of 150 spring barley varieties was investigated under artificial and natural infection conditions in the field. / Panaudojant molekulinius žymeklius ištirta tinkliškosios dryžligės patogeno Pyrenophora teres populiacijos genetinė įvairovė, nustatyti P. teres formų ir lytinio dauginimosi tipų sutinkamumas Lietuvoje, Latvijoje ir Estijoje. Lietuvos sąlygomis įvertintas Vakarų Europos ekotipo 150 vasarinių miežių veislių ir linijų jaurumas tinkliškajai dryžligei esant skirtingai pradinei infekcijai.

Made available in DSpace on 2016-12-06T17:42:04Z (GMT). No. of bitstreams: 1 PGPV14DA002.pdf: 382284 bytes, checksum: 85c7339f46122a0ce3079cb0b1db20fe (MD5) Previous issue date: 2014-04-28 / Brown spot (Bipolaris sorokiniana) and net blotch (Drechslera teres) are the main foliar diseases of barley in southern of Brazil. The objectives of this study were to evaluate the survival and viability of B. sorokiniana and D. teres on barley seeds during the off seasons and verify the influence of different barley seed treatments on: a. The fungi transmission to plants; b. The population of emerged plants; c. The brown spot and net-blotch intensity; d. The productivity; e. The seed health and f. the number of captured spores in the air. Barley seeds of six cultivars from four regions were used for viability analysis. Seed sanity tests were developed during ten months. Samples of 400 seeds were disinfected by sodium hypochlorite (4%) and whashed by sterile distilled water. The samples were plated in the Potato Dextrose Agar medium and incubated in growth chamber for seven to ten days. Field experiments were carried out with two barley cultivars sowed in two different dates of 2012 and 2013 growing season. The experiments were performed with treatments, control (without fungicide seed treatment), commercial treatment and additional laboratory treatments. The experiments were conducted in a randomized block design with four replications. The number of symptomatic plants with brown spot and net-blotch diseases was assessed from five to seven days intervals during 40 days. The incidence and severity foliar were quantified from 40 to 95 days after sowing in ten tillers randomly per plot. Collectors spores like windmill with microscope slide smeared with a mixture of phenol + hexane + Vaseline + paraffin were installed in the field. The microscope slides were changed weekly, and the collectors remained in the field up to the 38 plant ear emergence. Grain yield, grain classification and thousand kernels was assessed during the harvest and the seeds submitted to pathology test. The incidence and viability of B. sorokiniana and D. teres reduced with the storage. The viability average reduction of B. sorokiniana and D. teres was 27% and 30% in the off season, respectively. None of the simultaneus seed treatments eradicated the fungi. Seed treatments allowed fungi transmission to the plant leaves. Seeds commercial treatment was not effective in the fungi eradication, allowing up to 90% transmission to plants. Additional seed treatments reduced up tp 89% the fungi transmission. Commercial seed treatment showed the AUDPCS of 519.0 and 139.0 for net blotch and brown spot, respectively. The most efficient seed treatment was triadimenol + difenoconazol + carbendazim + thiamethoxam, reducing the B. sorokiniana and D. teres AUSPC in 11.4 and 120.5, respectively. The highest fungi conidia capturing occurred in field under commercial treatment seeds. There was a positive and significant correlation (r = 0.89 B. sorokiniana and r = 0.70 D. teres) between the number of spores in the air and severity. Seed treatment influenced the sanitary quality of barley seeds. There is a significant and positive correlation (r = 0.99) between the brown spot and net-blotch AUDPC and the incidence of B. sorokiniana and D. teres in the harvested seed. Commercial seed treatment did not reduce the B. sorokiniana and D. teres inocula in barley seeds. Additional, commercial seed treatment anticipated the begining of brown spot and net blotch epidemic, increasing yield costs. All other treatments did not eradicate the fungi. However, they delayed the bigining of diseases, enabling the farmer profit increase / A mancha-marrom (Bipolaris sorokiniana) e a mancha-em-rede (Drechslera teres) são as principais doenças fúngicas foliares da cevada no sul do Brasil. Os objetivos foram: quantificar a sobrevivência e a viabilidade de B. sorokiniana e D. teres em sementes de cevada durante a entressafra e verificar o efeito de diferentes tratamentos de sementes de cevada na transmissão dos fungos para a parte aérea das plantas, na população de plantas emersas, na intensidade da mancha marrom e da mancha-em-rede, na produtividade, na sanidade de sementes colhidas e no número de conídios capturados no ar. Foram utilizadas sementes de cevada de seis cultivares oriundas de quatro regiões para a análise de viabilidade. Os testes de sanidade foram desenvolvidos durante dez meses. Amostras de 400 sementes foram desinfestadas em hipoclorito de sódio (4%) e água destilada esterilizada, distribuídas em meio de cultura Batata-Dextrose-Ágar e encubadas em câmara de crescimento durante sete a dez dias. Os experimentos de campo foram conduzidos em 2012 e 2013 em duas épocas de semeadura e duas cultivares. Os tratamentos utilizados foram: testemunha (sem tratamento fungicida de sementes), tratamento comercial e tratamentos adicionais testados em laboratório. O modelo experimental foi em blocos casualisados e quatro repetições. O número de plantas sintomáticas com mancha marrom e mancha-em-rede foi quantificado em intervalos de cinco a sete dias até 40 dias após a semeadura. A incidência e a severidade foliar foram quantificadas desde os 40 até os 95 dias após a semeadura em dez perfilhos coletados ao acaso de cada parcela. Foram instalados coletores de esporos tipo cata-vento contendo lâmina de microscopia untada com mistura de fenol+hexano+vaselina+parafina. As lâminas foram trocadas semanalmente, e os coletores permaneceram no campo até o espigamento das plantas. Na colheita, foi quantificado rendimento de grãos, classificação e massa de mil grãos. As sementes colhidas foram submetidas ao teste de sanidade de sementes. Houve redução da incidência e da viabilidade de B. sorokiniana e D. teres com o armazenamento. A redução média viabilidade de B. sorokiniana e D. teres foi de 27% e 30% na entressafra, respectivamente. Nenhum dos tratamentos erradicou os fungos simultaneamente das sementes e possibilitaram transmissão para a parte aérea as plantas. O tratamento comercial de sementes não foi eficiente na erradicação dos patógenos com transmissão para a parte aérea de até 90%. Tratamentos de sementes adicionais reduziram a transmissão dos fungos em até 89%. O tratamento comercial de sementes antecipou as doenças foliares com AACPS de até 519,0 de mancha em rede e 139,0 de mancha marrom. O tratamento de semente mais eficiente (triadimenol + difenoconazol + carbendazim + tiametoxan) reduziu a AACPS das doenças em 11,4 e 120,5, respectivamente. A maior captura de conídios no ar dos fungos ocorreu nas plantas submetidas ao tratamento comercial de sementes havendo correlação positiva e significativa (r = 0,89 B. sorokiniana e r = 0,70 D. teres) entre número de conídios no ar e severidade das doenças. O tratamento de semente utilizado influenciou a qualidade sanitária das sementes de cevada produzidas, com correlação significativa e positiva (r=0,99) entre AACPS da mancha marrom e da mancha-em-rede e incidência de B. sorokiniana e D. teres nas sementes colhidas. O tratamento comercial de sementes não reduziu o inóculo de B. sorokiniana e D. teres das sementes de cevada, antecipa a epidemia da mancha marrom e mancha-em-rede e aumenta custo de produção. Os demais tratamentos apesar de não erradicar os fungos, retardam as doenças e aumentam o lucro do agricultor