Spot Form Of Net Blotch Research Articles

Barley disease recognition using deep neural networks

Plant disease negatively impacts food production and quality. It is crucial to detect and recognise plant diseases correctly. Traditional approaches do not offer a rapid and comprehensive management system for detecting plant diseases. Deep learning techniques (DL) have achieved encouraging results in discriminating patterns and anomalies in visual samples. This ability provides an effective method to diagnose any plant disease symptoms automatically. However, one of the limitations of recent studies is that in-field disease detection is underexplored, so developing a model that performs well for in-field samples is necessary. The objective of this study is to develop and investigate DL techniques for in-field disease detection of barley (Hordeum vulgare L.), one of the main crops in Australia, given visual samples captured at barley trials using a consumer-grade RGB camera. Consequently, A dataset was captured from test-bed trials across multiple paddocks infected with three diseases: net form net blotch (NFNB), spot form net blotch (SFNB), and scald, in various weather conditions. The collected data, 312 images (6000 × 4000 pixels), are divided into patches of 448 × 448 pixels, which are manually annotated into four classes: no-disease, scald, NFNB and SFNB. Finally, the data was augmented using random rotation and flip to increase the dataset size. The generated barley disease dataset is then applied to several well-known pre-trained DL networks such as DenseNet, ResNet, InceptionV3, Xception, and MobileNet as the network backbone. Given limited data, these methods can be trained to detect anomalies in visual samples. The results show that MobileNet, Xception, and InceptionV3 performed well in barley disease detection. On the other hand, ResNet showed poor classification ability. Moreover, Augmenting the data improves the performance of DL networks, particularly for underperforming backbones like ResNet, and mitigates the limited data access for these data-intensive networks. The augmentation step improved MobileNet performance by approximately 6 %. MobileNet achieved the highest accuracy of 98.63 % (the average of the three diseases) in binary classification and an accuracy of 93.50 % in multi-class classification. Even though classifying SFNB and NFNB is challenging in the early stages, MobileNet achieved the minimum misclassification rate among the two diseases. The results show the efficiency of this model in diagnosing barley diseases using complex data collected from the field environment. In addition, the model is lighter and comprises fewer trainable parameters. Consequently, MobileNet is suitable for small training datasets, reducing data acquisition costs.

High resolution mapping of a novel non-transgressive hybrid susceptibility locus in barley exploited by P. teres f. maculata

Hybrid genotypes can provide significant yield gains over conventional inbred varieties due to heterosis or hybrid vigor. However, hybrids can also display unintended negative attributes or phenotypes such as extreme pathogen susceptibility. The necrotrophic pathogen Pyrenophora teres f. maculata (Ptm) causes spot form net blotch, which has caused significant yield losses to barley worldwide. Here, we report on a non-transgressive hybrid susceptibility locus in barley identified between the three parental lines CI5791, Tifang and Golden Promise that are resistant to Ptm isolate 13IM.3. However, F2 progeny from CI5791 × Tifang and CI5791 × Golden Promise crosses exhibited extreme susceptibility. The susceptible phenotype segregated in a ratio of 1 resistant:1 susceptible representing a genetic segregation ratio of 1 parental (res):2 heterozygous (sus):1 parental (res) suggesting a single hybrid susceptibility locus. Genetic mapping using a total of 715 CI5791 × Tifang F2 individuals (1430 recombinant gametes) and 149 targeted SNPs delimited the hybrid susceptibility locus designated Susceptibility to Pyrenophora teres 2 (Spt2) to an ~ 198 kb region on chromosome 5H of the Morex V3 reference assembly. This single locus was independently mapped with 83 CI5791 × Golden Promise F2 individuals (166 recombinant gametes) and 180 genome wide SNPs that colocalized to the same Spt2 locus. The CI5791 genome was sequenced using PacBio Continuous Long Read technology and comparative analysis between CI5791 and the publicly available Golden Promise genome assembly determined that the delimited region contained a single high confidence Spt2 candidate gene predicted to encode a pentatricopeptide repeat-containing protein.

Global Spread, Genetic Differentiation, and Selection of Barley Spot Form Net Blotch Isolates.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread worldwide in the twentieth century. Genetic relationships were analyzed to determine the diversity, survival, and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor, and Europe. The results, based on genome-wide DArTseq data, indicated that isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged, and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counterselection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Do Growers Using Solo Fungicides Affect the Durability of Disease Control of Growers Using Mixtures and Alternations? The Case of Spot-Form Net Blotch in Western Australia.

Growers often use alternations or mixtures of fungicides to slow down the development of resistance to fungicides. However, within a landscape, some growers will implement such resistance management methods, whereas others do not, and may even apply solo components of the resistance management program. We investigated whether growers using solo components of resistant management programs affect the durability of disease control in fields of those who implement fungicide resistance management. We developed a spatially implicit semidiscrete epidemiological model for the development of fungicide resistance. The model simulates the development of epidemics of spot-form net blotch disease, caused by the pathogen Pyrenophora teres f. maculata. The landscape comprises three types of fields, grouped according to their treatment program, with spore dispersal between fields early in the cropping season. In one field type, a fungicide resistance management method is implemented, whereas in the two others, it is not, with one of these field types using a component of the fungicide resistance management program. The output of the model suggests that the use of component fungicides does affect the durability of disease control for growers using resistance management programs. The magnitude of the effect depends on the characteristics of the pathosystem, the degree of inoculum mixing between fields, and the resistance management program being used. Additionally, although increasing the amount of the solo component in the landscape generally decreases the lifespan within which the resistance management program provides effective control, situations exist where the lifespan may be minimized at intermediate levels of the solo component fungicide. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Field Evaluation of Fungicides and Host Plant Resistance Against Spot Form of Net Blotch (Pyrenophora Teres F. Maculata) Disease of Barley in South Tigray, Ethiopia

Field Evaluation of Fungicides and Host Plant Resistance Against Spot Form of Net Blotch (Pyrenophora Teres F. Maculata) Disease of Barley in South Tigray, Ethiopia

Major Susceptibility Gene Epistasis over Minor Gene Resistance to Spot Form Net Blotch in a Commercial Barley Cultivar.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant global disease of barley (Hordeum vulgare). Baudin, a barley cultivar that was until recently extensively grown in Western Australia, was reported as having minor seedling resistance. However, Baudin was highly susceptible to a local isolate, M3, suggesting that this isolate had gained virulence against a major susceptibility gene. M3 causes atypical lesions with pale centers early in the infection, with initial screens of a segregating population indicating that this was determined by a single locus in the Baudin genome. The susceptibility was semidominant in F1 progeny and the susceptibility gene, designated Spm1 (Susceptibility to P. teres f. maculata 1), mapped to a 190-kb section of the resistance gene-rich Mla region of chromosome 1H. Phenotyping with Ptm SP1, a non-M3 pathotype, identified a seedling resistance locus on 2H. Minor gene resistance is generally regarded as potentially durable, but our findings suggest the resistance to spot form net blotch in Baudin is nullified by strong susceptibility conferred by a separate locus on 1H. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley

Key messageGenetic characterization of a major spot form net blotch susceptibility locus to using linkage mapping to identify a candidate gene and user-friendly markers in barley.Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is an economically important foliar diseases in barley. Although various resistance loci have been identified, breeding for SFNB-resistant varieties has been hampered due to the complex virulence profile of Ptm populations. One resistance locus in the host may be effective against one specific isolate, but it may confer susceptibility to other isolates. A major susceptibility QTL on chromosome 7H, named Sptm1, was consistently identified in many studies. In the present study, we conduct fine mapping to localize Sptm1 with high resolution. A segregating population was developed from selected F2 progenies of the cross Tradition (S) × PI 67381 (R), in which the disease phenotype was determined by the Sptm1 locus alone. Disease phenotypes of critical recombinants were confirmed in the following two consecutive generations. Genetic mapping anchored the Sptm1 gene to an ⁓400 kb region on chromosome 7H. Gene prediction and annotation identified six protein-coding genes in the delimited Sptm1 region, and the gene encoding a putative cold-responsive protein kinase was selected as a strong candidate. Therefore, providing fine localization and candidate of Sptm1 for functional validation, our study will facilitate the understanding of susceptibility mechanism underlying the barley-Ptm interaction and offers a potential target for gene editing to develop valuable materials with broad-spectrum resistance to SFNB.

Adjusting fungicide treatment programmes when resistance is developing: The case of spot‐form net‐blotch in Western Australia

AbstractWe use the case of spot‐form net‐blotch (SFNB) and azole fungicides in Western Australia to investigate the financial implications of a switch from fungicide A, to which high resistance is developing, to a more expensive fungicide B, to which less resistance has developed. This is done using a budget calculation coupled with an epidemiological model. The model calculates the total financial cost of disease as the sum of the expense of the fungicide treatment programme and the cost of disease‐induced yield loss. The optimal treatment programme is that which minimizes the total cost of disease. We show that when the pathogen population is dominated by wild‐type strains, propiconazole is the fungicide minimizing the cost of disease. The number of applications and optimal dose depend on the potential yield of the field under consideration. When the pathogen population is dominated by a strain highly resistant to propiconazole, the use of this fungicide will not give a positive financial return. In this case the azole mixture, prothioconazole + tebuconazole is more cost effective. The pathogen strains with medium resistance to propiconazole have little effect on the optimal treatment programme compared to the wild type. We also use the model to calculate, depending on the potential yield and on whether an effective seed treatment is used, the frequency of the highly resistant strains at which the switch from the use of propiconazole to the use of prothioconazole + tebuconazole is economically justified.

Widespread genetic heterogeneity and genotypic grouping associated with fungicide resistance among barley spot form net blotch isolates in Australia.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a major foliar disease of barley worldwide. Knowledge of the pathogen's genetic diversity and population structure is critical for a better understanding of inherent evolutionary capacity and for the development of sustainable disease management strategies. Genome-wide, single nucleotide polymorphism data of 254 Australian isolates revealed genotypic diversity and an absence of population structure, either between states, or between fields and cultivars in different agro-ecological zones. This indicates there is little geographical isolation or cultivar directional selection and that the pathogen is highly mobile across the continent. However, two cryptic genotypic groups were found only in Western Australia, predominantly associated with genes involved in fungicide resistance. The findings in this study are discussed in the context of current cultivar resistance and the pathogen's adaptive potential.

Identification of novel and stable genomic regions associated with barley resistance to spot form net blotch disease under different temperature conditions during the reproductive stage

AbstractSpot form net blotch (SFNB) is one of the most important barley diseases in the world; abiotic stresses caused by climate change are also a problem for cultivation of cereals, including barley, on a global level. Identifying stable genomic regions associated with resistance to SFNB in different temperature conditions at the reproductive stage can accelerate the process of producing new cultivars resistant to this disease. In this study, 103 F8 lines, resulting from the cross of cultivars Kavir and Badia, were cultivated in three years on three different dates. After artificial inoculation with Pyrenophora teres f. maculata Gonbad Kavous isolate, the plants were evaluated at the adult stage. The planting dates were chosen so that during early, conventional and delayed planting dates, the reproductive stage of the lines coincided with cold, non‐thermal and heat stress conditions, respectively. A genome‐wide composite interval mapping method was used to identify quantitative trait loci (QTLs) controlling SFNB and estimate the effect of each of them. In the cold, non‐thermal and heat stress conditions at the reproductive stage, three (three major), four (two major) and six (one major) stable resistance QTLs against SFNB were detected, respectively. The stable QTLs of resistance including QRPtma‐2H, QRPtma‐3H and QRPtma‐7H were located on chromosomes 2H, 3H and 7H, respectively. After flanking markers confirm the resistance of QTLs to SFNB, the SCoT7‐C, ISJ17‐B and Bmac0144k markers can be used in the screening resistance of cultivars under non‐thermal and heat stress conditions. Moreover, the Bmac0282b and Ebmatc0016 markers can be used for screening resistance of cultivars under cold stress conditions in marker‐assisted selection projects.

Increased levels of cell wall degrading enzymes and peptidases are associated with aggressiveness in a virulent isolate of Pyrenophora teres f. maculata

Pyrenophora teres f. maculata (Ptm) is a fungal pathogen that causes the spot form of net blotch on barley and leads to economic losses in many of the world's barley-growing regions. Isolates of Ptm exhibit varying levels of aggressiveness that result in quantifiable changes in the severity of the disease. Previous research on plant-pathogen interactions has shown that such divergence is reflected in the proteome and secretome of the pathogen, with certain classes of proteins more prominent in aggressive isolates. Here we have made a detailed comparative analysis of the secretomes of two Ptm isolates, GPS79 and E35 (highly and mildly aggressive, respectively) using a proteomics-based approach. The secretomes were obtained in vitro using media amended with barley leaf sections. Secreted proteins therein were harvested, digested with trypsin, and fractionated offline by HPLC prior to LC-MS in a high-resolution instrument to obtain deep coverage of the proteome. The subsequent analysis used a label-free quantitative proteomics approach with relative quantification of proteins based on precursor ion intensities. A total of 1175 proteins were identified, 931 from Ptm and 244 from barley. Further analysis revealed 160 differentially abundant proteins with at least a two-fold abundance difference between the isolates, with the most enriched in the aggressive GPS79 secretome. These proteins were mainly cell-wall (carbohydrate) degrading enzymes and peptidases, with some oxidoreductases and other pathogenesis-related proteins also identified, suggesting that aggressiveness is associated with an improved ability of GPS79 to overcome cell wall barriers and neutralize host defense responses.

Host and pathogen genetics reveal an inverse gene-for-gene association in the P. teres f. maculata-barley pathosystem.

Pathogen and host genetics were used to uncover an inverse gene-for-gene interaction where virulence genes from the pathogen Pyrenophora teres f. maculata target barley susceptibility genes, resulting in disease. Although models have been proposed to broadly explain how plants and pathogens interact and coevolve, each interaction evolves independently, resulting in various scenarios of host manipulation and plant defense. Spot form net blotch is a foliar disease of barley caused by Pyrenophora teres f. maculata. We developed a barley population (Hockett × PI 67381) segregating for resistance to a diverse set of P. teres f. maculata isolates. Quantitative trait locus analysis identified major loci on barley chromosomes (Chr) 2H and 7H associated with resistance/susceptibility. Subsequently, we used avirulent and virulent P. teres f. maculata isolates to develop a pathogen population, identifying two major virulence loci located on Chr1 and Chr2. To further characterize this host-pathogen interaction, progeny from the pathogen population harboring virulence alleles at either the Chr1 or Chr2 locus was phenotyped on the Hockett × PI 67381 population. Progeny harboring only the Chr1 virulence allele lost the barley Chr7H association but maintained the 2H association. Conversely, isolates harboring only the Chr2 virulence allele lost the barley Chr2H association but maintained the 7H association. Hockett × PI 67381 F2 individuals showed susceptible/resistant ratios not significantly different than 15:1 and results from F2 inoculations using the single virulence genotypes were not significantly different from a 3:1 (S:R) ratio, indicating two dominant susceptibility genes. Collectively, this work shows that P. teres f. maculata virulence alleles at the Chr1 and Chr2 loci are targeting the barley 2H and 7H susceptibility alleles in an inverse gene-for-gene manner to facilitate colonization.

Using a Hybrid Mapping Population to Identify Genomic Regions of Pyrenophora teres Associated With Virulence.

Net blotches caused by Pyrenophora teres are important foliar fungal diseases of barley and result in significant yield losses of up to 40%. The two types of net blotch, net-form net blotch and spot-form net blotch, are caused by P. teres f. teres (Ptt) and P. teres f. maculata (Ptm), respectively. This study is the first to use a cross between Ptt and Ptm to identify quantitative trait loci (QTL) associated with virulence and leaf symptoms. A genetic map consisting of 1,965 Diversity Arrays Technology (DArT) markers was constructed using 351 progenies of the Ptt/Ptm cross. Eight barley cultivars showing differential reactions to the parental isolates were used to phenotype the hybrid progeny isolates. Five QTL associated with virulence and four QTL associated with leaf symptoms were identified across five linkage groups. Phenotypic variation explained by these QTL ranged from 6 to 16%. Further phenotyping of selected progeny isolates on 12 more barley cultivars revealed that three progeny isolates are moderately to highly virulent across these cultivars. The results of this study suggest that accumulation of QTL in hybrid isolates can result in enhanced virulence.

Investigating InVitro Mating Preference Between or Within the Two Forms of Pyrenophora teres and Its Hybrids.

Net blotch diseases result in significant yield losses to barley industries worldwide. They occur as net-form and spot-form net blotch caused by Pyrenophora teres f. teres and P. teres f. maculata, respectively. Hybridization between the forms was proposed to be rare, but recent identifications of field hybrids has renewed interest in the frequency and mechanisms underlying hybridization. This study investigates the mating preference of P. teres f. teres, P. teres f. maculata, and laboratory-produced hybrids invitro, using 24 different isolates and four different experimental setups. Two crosses in our study produced ascospores during two intervals separated by a 32- to 35-day period of no ascospore production. For these crosses, P. teres f. teres isolates mated with isolates of the same form during the early ascospore production interval, and produced hybrids during the later interval. P. teres f. maculata isolates did not mate with isolates of the same form, but instead hybridized with P. teres f. teres isolates. Analyses based on DArTseq markers confirmed that laboratory-produced hybrids, when given the choice to mate with both P. teres f. teres and P. teres f. maculata, mated with P. teres f. teres isolates. These results unravel a novel concept that P. teres f. teres seems to have a greater reproduction vigor than P. teres f. maculata, which could lead to increased prevalence of hybrid incidences invivo.

Efficacy of spot form of net blotch suppression in barley from seed, fertiliser and foliar applied fungicides

Spot form of net blotch (SFNB), caused by Pyrenophora teres f. maculata is a prevalent and damaging foliar disease of barley (Hordeum vulgare). Fungicides are commonly used to manage SFNB infection and reduce production loss in susceptible varieties. This study determined the efficacy of suppression of SFNB and associated grain yield and quality improvements from application of fungicides to seed, fertiliser and/or foliar treatments. Eight field experiments were conducted in south-eastern Australia during 2016–2018. Two experiments compared single applications of different commercially available seed, fertiliser and foliar fungicides. Six experiments evaluated different growth stage application timings of either a single application or combinations of seed applied fluxapyroxad and foliar applied prothioconazole + tebuconazole. Spot form of net blotch caused grain yield loss of up to 1.2 t/ha (20%), reduced grain retention (percentage seed width greater than 2.5 mm) and weight by up to 15% and 13%, respectively, and increased screenings (percentage seed width less than 2.2 mm) by up to 18%. Seed applied fluxapyroxad was the most effective fungicide treatment providing the greatest SFNB suppression, grain yield and quality increase of the single fungicide applications. The combination of seed applied fluxapyroxad with a foliar application of prothioconazole + tebuconazole at flag leaf emergence significantly increased grain yield and quality further in one experiment. The dual foliar fungicide application of prothioconazole + tebuconazole at stem elongation (Z31) and flag leaf emergence (Z39) was not as effective in providing SFNB suppression according to AUDPC but provided comparable grain yield and quality to the seed and foliar treatment. Of the foliar fungicides, prothioconazole + tebuconazole and bixafen + prothioconazole provided the best SFNB suppression, grain yield and quality. These fungicide applications strategies provide improved SFNB management options for barley growers, particularly in high yielding crops where grain yield exceeds 5 t/ha.

Genome-wide association mapping of Pyrenophora teres f. maculata and Pyrenophora teres f. teres resistance loci utilizing natural Turkish wild and landrace barley populations.

Unimproved landraces and wild relatives of crops are sources of genetic diversity that were lost post domestication in modern breeding programs. To tap into this rich resource, genome-wide association studies in large plant genomes have enabled the rapid genetic characterization of desired traits from natural landrace and wild populations. Wild barley (Hordeum spontaneum), the progenitor of domesticated barley (Hordeum vulgare), is dispersed across Asia and North Africa, and has co-evolved with the ascomycetous fungal pathogens Pyrenophora teres f. teres and P. teres f. maculata, the causal agents of the diseases net form of net blotch and spot form of net blotch, respectively. Thus, these wild and local adapted barley landraces from the region of origin of both the host and pathogen represent a diverse gene pool to identify new sources of resistance, due to millions of years of co-evolution. The barley—P. teres pathosystem is governed by complex genetic interactions with dominant, recessive, and incomplete resistances and susceptibilities, with many isolate-specific interactions. Here, we provide the first genome-wide association study of wild and landrace barley from the Fertile Crescent for resistance to both forms of P. teres. A total of 14 loci, four against P. teres f. maculata and 10 against P. teres f. teres, were identified in both wild and landrace populations, showing that both are genetic reservoirs for novel sources of resistance. We also highlight the importance of using multiple algorithms to both identify and validate additional loci.

The Occurrence of Barley (Hordeum vulgare) and Wild Barley (H. spontaneum) Leaf Diseases in Batman Province and Surrounding Areas of Turkey

Barley ( Hordeum vulgare ) is an important crop in Turkey. In this study, 37 barley fields in Batman central district and Beşiri (Batman), Gercüş (Batman), Hasankeyf (Batman), Kozluk (Batman), Sason (Batman), Kocaköy (Diyarbakır), Kurtalan (Siirt), and Midyat (Mardin) districts were examined for the presence of leaf diseases. In addition, 24 naturally grown wild barley ( H. spontaneum ) populations in Batman central district and Beşiri, Hasankeyf, Kurtalan and Midyat districts were inspected for the presence of leaf diseases. In barley fields, scald caused by the Rhynchosporium commune was the most common diseases followed by spot form of net blotch caused by Drechslera teres f. maculata , barley stripe caused by Drechslera graminea , net form of net blotch caused by Drechslera teres f. teres , spot blotch caused by Cochliobolus sativus , powdery mildew caused by Blumeria graminis f. sp. hordei and brown rust caused by Puccinia hordei . Among the Hordeum spontaneum populations the most common disease was scald, followed by spot form of net blotch, net form of net blotch, powdery mildew, and brown rust.

Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata

The fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (SFNB), is currently the most significant disease of barley in Australia and a major disease worldwide. Management of SFNB relies heavily on fungicides and in Australia the demethylase inhibitors (DMIs) predominate. There have been sporadic reports of resistance to DMIs in Ptm but the mechanisms remain obscure. Ptm isolates collected from 1996 to 2019 in Western Australia were tested for fungicide sensitivity levels. Decreased sensitivity to DMIs was observed in isolates collected after 2015. Resistance factors to tebuconazole fell into two classes; moderate resistance (MR; RF 6–11) and high resistance (HR; RFs 30–65). Mutations linked to resistance were detected in the promoter region and coding sequence of the DMI target gene Cyp51A. Solo-LTR insertion elements were found at 5 different locations in the promoter region. Three different non-synonymous mutations encoded an altered protein with a phenylalanine to leucine substitution at position 489, F489L (F495I in the archetype CYP51A of Aspergillus fumigatus). F489L mutations have also been found in DMI-resistant strains of P. teres f. sp. teres. Ptm isolates carrying either a LTR insertion element or a F489L allele displayed the MR1 or MR2 phenotypes, respectively. Isolates carrying both an insertion element and a F489L mutation displayed the HR phenotype. Multiple mechanisms acting both alone and in concert were found to contribute to DMI resistance in Ptm. Moreover, these mutations have emerged repeatedly in Western Australian Ptm populations by a process of parallel evolution.

First Report of Spot Blotch of Barley Caused by Cochliobolus sativus in Morocco

Barley (Hordeum vulgare L.) is grown on ∼2 million hectares in Morocco (av. 1.2 t/ha), with ∼70% under semiarid conditions (FAO 2017). Historically, net form net blotch (NFNB) (Pyrenophora teres f. teres [Ptt]) and spot form net blotch (SFNB) (P. teres f. maculata [Ptm]) have predominated in Morocco, causing up to 29% yield losses (El Yousfi and Ezzahiri 2002). Often, barley spot blotch (SB) caused by Cochliobolus sativus (anamorph: Bipolaris sorokiniana [Sacc.] Shoem.) is misdiagnosed as SFNB. Disease surveys were conducted in six regions (Abda, Rommani, Choiya, Dokhala, Gharb, and Rahamna) in 2015 (54 fields) and 2018 (49 fields) at heading stage and covered three cultivars (Annoucer, Rabat 071, and Arig 08) representing 34, 41, and 25% of fields, respectively. Leaf samples (∼10) from three plants per field were collected. From each site, a representative sample with SFNB symptoms (20 leaf segments, 1-cm² pieces) was used for isolation. Samples were surface sterilized with 50% ethanol for 30 s and 5% bleach for 60 s, followed by rinsing in sterile deionized water twice for 5 min. Samples were dried between two layers of sterile filter paper, followed by incubation on PDA under 12 h light/dark at 22 ± 1°C. After 4 to 5 days, dark brown elliptical conidia of 60 to 120 × 12 to 20 µm with 5 to 9 septa per cell were observed. Cultures sporulated profusely with light to dark gray or brown cottony mass with wavy colony margins (Kumar et al. 2002). Based on morphology, isolates were identified as C. sativus. Of 103 samples, C. sativus was recovered from 11 (11%), and 92 samples were Ptt (40%) and Ptm (50%). To test pathogenicity of the SB isolates, two SB differential genotypes (NDB5883 and Bowman) were used as checks, along with three cultivars (Rabat 071, Annoucer, and Oussama). Seeds (∼4 to 5 per genotype per replicate) were sown in three replications in peat moss with 14-14-14 NPK; seedlings were raised in a growth chamber with 16 h light/8 h dark at 20 ± 1°C. To produce inoculum, lyophilized agar plugs of monoconidial SB isolates were incubated on V8PDA in the dark for 4 to 5 days at 22 ± 1°C, followed by 22 ± 1°C with 12 h light/dark for 7 to 8 days. V8PDA plates were flooded with 5 to 10 ml of sterile distilled water, and the conidia were harvested followed by filtration with a double layer of cheese cloth. Spore density was adjusted to 5,000 conidia/ml supplemented with surfactant (0.01% Tween 20). Seedlings at the two-leaf stage (∼14 days old) were inoculated with the two isolates independently until runoff and incubated at 100% RH at 20 ± 1°C in the dark for 24 h. Seedlings were transferred to a greenhouse with 16 h light/8 h dark at 20 ± 1°C. After 10 days, the seedlings were scored on a 0 to 9 scale (Fetch and Steffenson 1999). All infected seedlings produced lesions as described by Kumar et al. (2002) and Fetch and Steffenson (1999). The experiment was carried out twice to confirm the repeatability of SB symptoms. To satisfy Koch’s postulates, leaf tissue from infected seedlings of all genotypes for both SB isolates was surface sterilized and incubated on PDA as described above. Monoconidial cultures were induced for sporulation on V8PDA; upon inoculation, similar symptoms were observed, confirming SB. Both SB isolates were grown in Fries medium, and genomic DNA was isolated (Benslimane et al. 2013). PCR with ITS1-F/ITS2-R produced a single band of ∼500 bp and was subjected to DNA sequencing from both ends. Homology-based BLASTN searches using sequences from both SB isolates produced 100% identity (E-value 0) to 98 GenBank accessions of SB partial sequence of small and large subunit rRNA, complete sequence of ITS1, 5.8S rRNA gene, and ITS2 (MK676001, MK905507, MH864698). Conidia and colony morphology, symptomology, Koch’s postulates, and DNA sequencing corroborate C. sativus (perfect stage) causing SB in barley. The queried sequences were deposited in GenBank as MN444781, 82, 84, and 85. This is the first report of SB in Morocco. This has implications for future barley breeding and cultivation under dry areas, as SB so far has been a disease of hot and humid conditions of South Asia.

Mapping of barley susceptibility/resistance QTL against spot form net blotch caused by Pyrenophora teres f. maculata using RIL populations.

Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an important disease of barley worldwide including the major barley production regions of North America. To characterize SFNB resistance/susceptibility quantitative trait loci (QTL), three recombinant inbred line (RIL) populations were developed from crosses between the malting barley cultivars, Tradition (six row) and Pinnacle (two row), and the two world barley core collection lines, PI67381 and PI84314. Tradition and Pinnacle were susceptible to many North American Ptm isolates, while PI67381 and PI84314 carry resistances to diverse Ptm isolates from across the globe. The RIL populations, Tradition/PI67381, Pinnacle/PI67381, and Pinnacle/PI84314 were genotyped using polymerase chain reaction-mediated genotype-by-sequencing single nucleotide polymorphism marker panels and phenotyped at the seedling stage with six geographically distinct Ptm isolates: FGOB10Ptm-1 (North Dakota, USA), Pin-A14 (Montana, USA), Cel-A17 (Montana, USA), SG1 (Australia), NZKF2 (New Zealand) and DEN2.6 (Denmark). The goal was to determine if the susceptible elite lines contained common susceptibility genes/QTL or if the resistant lines had common resistant genes/QTL effective against diverse Ptm isolates. The QTL analyses identified a total of 12 resistance and/or susceptibility loci on chromosomes 2H, 3H, 4H, 6H, and 7H of which three had not been previously reported. Common major QTL were detected on chromosome 2H (R2 = 14-40%) and 7H (R2 = 24-80%) in all three RIL populations, suggesting underlying genes with broad resistance specificity. The major 7H QTL was shown to be a dominant susceptibility gene in both susceptible malting barley varieties.