Spatial heterogeneity of disease infection attributable to neighbour genotypic identity in barley cultivars

The aims of this study were to identify resistance to spot form of net blotch (SFNB) in spring barley and to investigate the stability of SFNB resistance in Morocco. The seedling resistance to SFNB was evaluated by inoculating 340 barley genotypes with the Pyrenophora teres F. maculata (Ptm) isolate FGOB10Ptm-1 (FGO) in the greenhouse. The same barely genotypes were evaluated for adult-stage plant resistance to SFNB in field trials in Morocco. All experiments were conducted in alpha-lattices with two replicates. SFNB disease severity was estimated on barley leaves using double digit scale. To investigate the stability of both qualitative and quantitative resistance to SFNB, 104 barley genotypes were subjected to AMMI analysis. Differential responses of SFNB barley resistance to FGO were found at the seedling stage in the greenhouse. Twelve genotypes showing scores of <1.5, at the seedling stage, were determined to be highly resistant to FGO. The ANOVA showed highly significant (p < 0.001) effects of genotype (G) and G × E (E-Pathotypes of SFNB) interaction on SFNB severity among the 340 barley genotypes at the adult plant stage. The AMMI ANOVA showed that IPCA1, IPCA2 and IPCA3 accounted for 77.9% of the variation of the G × E interaction for SFNB severity. The G × E interaction consisted of divergent genotypic responses to SFNB severity due to different pathotypes prevalent in hot-spot environments. The AMMI stability value demonstrated that barley genotypes AM-14, 30, 31, 68, 107, 108, 112, 149, 170, 185, 204, 240, 304, 326, 326 and 337 were resistant and stable across hot-spot environments against SFNB. Divergent environmental responses of SFNB were recorded in MCH_2015, SE_2015, AT_2016, SE_2016 and JS_2015. Stable SFNB resistant genotypes are valuable resources for the introgression of qualitative and quantitative resistance to barley in Morocco.

The responses of 95 barley lines and cultivars to spot form of net blotch (SFNB) caused by Pyrenophora teres f. maculata were analyzed as seedlings and adults in Australia and Canada. Cluster analyses revealed complex reaction responses. Only 2 lines (Esperance Orge 289 and TR3189) were resistant to all isolates at the seedling stage, whereas 15 lines and cultivars (81-82/033, Arimont, BYDV-018, CBSS97M00855T-B2-M1-Y1-M2-Y-1M-0Y, CI9776, Keel, Sloop, Torrens, TR326, VB0111, Yarra, VB0229, WI-2477, WI2553, and Wisconsin Pedigree) were resistant toward the two Canadian isolates and mixture of Australian isolates at the adult stages. In Australian field experiments, the effectiveness of SFNB resistance in three barley cultivars (Barque, Cowabbie, and Schooner) and one breeding line (VB9104) with a different source of resistance was tested. Barque, which possessed a resistance gene that provided complete resistance to SFNB, was the most effective and showed no effect on grain yield or quality in the presence of inoculum. Generally, cultivars with seedling or adult resistance had less disease and better grain quality than the susceptible control, Dash, but they were not as effective as Barque. A preliminary differential set of 19 barley lines and cultivars for P. teres f. maculata is proposed.

Pyrenophora teres Drechs. causes net blotch of barley, a common foliar disease in cultivation zones around the world. The disease occurs in two forms, namely a net form net blotch (NFNB) caused by P. teres f. teres and a spot form net blotch (SFNB) caused by P. teres f. maculata. As in other parts of the northern Great Plains, in the Mon-Dak area (western North Dakota and eastern Montana), NFNB is prevalent. SFNB was first reported in western Montana in 1983 (1) and more recently in eastern North Dakota in 2010 (3) but not in the Mon-Dak area. In the summer of 2011, unusual spot lesions that were surrounded by necrosis or chlorosis were observed on different barley cultivars in fields at Williston, ND, Nesson Valley, ND, and Sidney, MT areas. Diseased leaves from various barley cvs. from the three locations were transferred to water agar and incubated at room temperature for 24 h to induce sporulation. Morphological examination of conidia (45 to 169 × 15 to 21 μm) did not show significant differences from a known isolate of P. teres f. teres 0-1 (provided by Tim Friesen, ARS, Fargo, ND). For pathogenicity testing, six 14-day-old plants of barley cv. Tradition were sprayed until runoff with a 2,000 spore/ml suspension of two isolates from each location and the control P. teres f. maculata isolate DEN2.6 (provided by Tim Friesen). Plants were incubated first in a lighted humidity chamber for 24 h and then in a greenhouse for 7 days at 21°C. Regardless of inoculum source, spot lesions surrounded by necrosis or chlorosis, typical of SFNB, appeared on the inoculated leaves within 7 days. Fungi isolated from symptomatic leaves were identified as P. teres and the morphology of the conidia was undistinguishable from those of P. teres f. teres. All control plants which were sprayed with sterile distilled water were symptomless. The pathogenicity test was repeated. Rapid PCR detection and amplicon sequencing (2) of the internal transcribed spacer (ITS) region of ribosomal genes was performed on field and pathogenicity test leaf lesion samples to confirm the presence of P. teres f. maculata. DNA templates were prepared using the Extract-N-Amp Plant PCR Kits (Sigma Chemical Co., St. Louis, MO) and subjected to PCR using ITS1 and ITS4 primers. Amplicons were then purified and sequenced. The 585-bp nucleotide sequences of P. teres f. maculata from Mon-Dak area were submitted to GenBank under accession nos. PtmNES1 (JX187587), PtmSDY1 (JX187588), PtmSDY2 (JX187589), and PtmWIL1 (JX187590). The sequences from the four locations shared 100% similarity and also with P. teres f. maculata (EF452471) from GenBank while showing 10 nucleotide differences (99% similarity) with P. teres f. teres (EF452472).The results represent first report of SFNB in the Mon-Dak. Barley is one of the most important crops in the area. Resistance of the NFNB and SFNB of barley are controlled by different genes (4). Based on this report, SFNB therefore have to be considered in selection of barley cultivars for cultivation in the area.

Spot form of net blotch (SFNB), caused by the fungus Pyrenophora teres f. maculata, was first described in Denmark in the 1960s and is now a prevalent foliar disease of barley in many countries. This disease should be controlled as a separate disease-causing organism from the net form of net blotch (NFNB), which is caused by P. teres f. teres. The increase in prevalence of SFNB is primarily due to stubble retention and cultivation of susceptible varieties, which have resulted in increased inoculum. Infected barley stubble is the primary inoculum source for SFNB, producing both asexual spores (conidia) and sexual spores (ascospores) from pseudothecia. Spot form of net blotch causes significant losses in grain yield and quality in situations where inoculum is present, susceptible varieties are cultivated, and where the climate is cool and moist. Cultivation of resistant varieties is the most cost-effective method for control of SFNB and more than 12 different resistance sources have been identified in barley germplasm and wild barley relatives. The resistance loci of 11 of these have been mapped. Control of SFNB can also be achieved with application of foliar fungicides, crop rotation, and stubble destruction.

Two forms of barley net blotch are caused by different formae of the fungus Pyrenophora teres and both are economically important pathogens. The spot form of the net blotch fungus (P. teres f. maculata) and the net form of the net blotch fungus (P. teres f. teres) cause the lesion types indicated by their disease names, although symptom overlap and similar spore morphology can make identification difficult. Randomly amplified polymorphic DNA fragments differentiated the two forms of Pyrenophora. Polymorphic bands were cloned and sequenced to develop specific primer sets. A simple assay based on the polymerase chain reaction was developed and can identify the Pyrenophora formae causing disease symptoms directly from infected plant tissues in a single multiplex reaction. The assay was validated using amplified fragment length polymorphism genotyping of isolates and was shown to be more accurate than reliance on symptom expression. This assay can now be used for routine diagnosis, epidemiological studies and resistance breeding, where correct identification of each pathogen is critical.

A recently developed spot form of blotch differential set of 16 barley lines was tested for reaction response to 60 Pyrenophora teres f. maculata isolates from geographically disperse barley crops of Australia. Twelve barley lines (Arimont, Barque, Chebec, CI5286, CI5791, CI9214, CII6150, Dairokkaku, Esperance Orge 289, Galleon, Keel, Skiff, Torrens and TR250) provided differential response between the isolates. The susceptible controls Gairdner and Kombar provided indication of isolate virulence or avirulence. Abundant pathogenic diversity was revealed with 33 designated pathotypes, some of which related to geographic region. AFLP analysis also revealed abundant diversity with each of the isolates representing a unique genotype and one isolate that contained both AFLP bands unique to P. teres f. maculata and P. teres f. teres, the cause of spot form and net form of net blotch respectively, suggesting that sexual recombination between the net form and spot form isolates may have occurred naturally in the field.

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 caused up to 25 % (0.9 t/ha) grain yield loss to barley and significant reductions in grain quality parameters such as retention (13 %), screenings (5 %) and grain weight (6 %) in the Wimmera region of Victoria during 2005–11. Spot form of net blotch was suppressed by varying degrees between experiments, seasons, and crop growth stages when foliar fungicide (propiconazole) was applied. Of single applications, treatment at the stem elongation (Z31) or flag leaf (Z39) growth stages generally provided the greatest disease suppression. Single application at tillering (Z25) or ear emergence (Z55) were generally ineffective. Eight chemical seed treatments registered for use in barley were tested as potential low cost management options for SFNB. Four seed treatments reduced SFNB severity in the glasshouse, but none were effective in the field.

Pyrenophora teres f. maculata, the causal agent of spot form of net blotch (SFNB), is an emerging pathogen of barley in the United States and Australia. Compared with net form of net blotch (NFNB), less is known in the U.S. Upper Midwest barley breeding programs about host resistance and quantitative trait loci (QTL) associated with SFNB in breeding lines. The main objective of this study was to identify QTL associated with SFNB resistance in the Upper Midwest two-rowed and six-rowed barley breeding programs using a genome-wide association study approach. A total of 376 breeding lines of barley were evaluated for SFNB resistance at the seedling stage in the greenhouse in Fargo in 2009. The lines were genotyped with 3,072 single nucleotide polymorphism (SNP) markers. Phenotypic evaluation showed a wide range of variability among populations from the four breeding programs and the two barley-row types. The two-rowed barley lines were more susceptible to SFNB than the six-rowed lines. Continuous distributions of SFNB severity indicate the quantitative nature of SFNB resistance. The mixed linear model (MLM) analysis, which included both population structure and kinship matrices, was used to identify significant SNP-SFNB associations. Principal component analysis was used to control false marker-trait association. The linkage disequilibrium (LD) estimates varied among chromosomes (10 to 20 cM). The MLM analysis identified 10 potential QTL in barley: SFNB-2H-8-10, SFNB-2H-38.03, SFNB-3H-58.64, SFNB-3H-78.53, SFNB-3H-91.88, SFNB-3H-117.1, SFNB-5H-155.3, SFNB-6H-5.4, SFNB-6H-33.74, and SFNB-7H-34.82. Among them, four QTL (SFNB-2H-8-10, SFNB-2H-38.03 SFNB-3H-78.53, and SFNB-3H-117.1) have not previously been published. Identification of SFNB resistant lines and QTL associated with SFNB resistance in this study will be useful in the development of barley genotypes with better SFNB resistance.

Surveys of incidence and severity of foliar diseases in a total of 127 barley (Hordeum vulgare) crops throughout Victoria were carried out during 2007 and 2008. In each crop, 100 tillers were assessed for disease symptoms on the top four leaves. Spot form of net blotch caused by Pyrenophora teres f. maculate had the highest incidence, being present in more than 90% of crops surveyed, with severity ranging from 0 to 42%. Scald caused by Rhynchosporium secalis had the second highest incidence, being found in 13 and 23% of crops in 2007 and 2008, respectively, with severity ranging from 0 to 16%. Leaf rust caused by Puccinia hordei was present in 13 and 18% of crops in 2007 and 2008, respectively, with severity of up to 3%. Powdery mildew caused by Erysiphe graminis was present in 11 and 9% of crops in 2007 and 2008, respectively, with severity below 0.1%. No other diseases, such as the net form of net blotch caused by P. teres f. teres were detected. The high incidence of spot form of net blotch, which can cause up to 44% grain yield loss, suggests that resistance traits need to be made a priority and targeted by barley breeding programs in Victoria for control of this important disease.

We report genomic regions that significantly control resistance to scald, net form (NFNB) and spot form net blotch (SFNB) in barley. Barley genotypes from Ethiopia, ICARDA, and the United States were evaluated in Ethiopia and North Dakota State University (NDSU). Genome-wide association studies (GWAS) were conducted using 23,549 single nucleotide polymorphism (SNP) markers for disease resistance in five environments in Ethiopia. For NFNB and SFNB, we assessed seedling resistance in a glasshouse at NDSU. A large proportion of the Ethiopian landraces and breeding genotypes were resistant to scald and NFNB. Most of genotypes resistant to SFNB were from NDSU. We identified 17, 26, 7, and 1 marker-trait associations (MTAs) for field-scored scald, field-scored net blotch, greenhouse-scored NFNB, and greenhouse-scored SFNB diseases, respectively. Using the genome sequence and the existing literature, we compared the MTAs with previously reported loci and genes for these diseases. For leaf scald, only a few of our MTAs overlap with previous reports. However, the MTAs found for field-scored net blotch as well as NFNB and SFNB mostly overlap with previous reports. We scanned the barley genome for identification of candidate genes within 250 kb of the MTAs, resulting in the identification of 307 barley genes for the 51 MTAs. Some of these genes are related to plant defense responses such as subtilisin-like protease, chalcone synthase, lipoxygenase, and defensin-like proteins.

Net blotch of barley (Hordeum vulgare L.) caused by the fungus Pyrenophora teres (anamorph Drechslera teres) is found in two forms, net form net blotch (NFNB) and spot form net blotch (SFNB). When inoculated on susceptible varieties, P. teres f. teres produces lesions with a characteristic net-like pattern surrounded by necrosis or chlorosis (NFNB), whereas P. teres f. maculata produces lesions consisting of spots surrounded by necrosis or chlorosis (SFNB). Recently, epidemics of SFNB have occurred throughout the world (4). Currently, net blotch is a significant foliar disease of barley in the North Dakota-Northwestern Minnesota agricultural region, a leading barley-production area. Diseased barley leaf tissue was collected annually from 2004 to 2008 in Fargo and Langdon, ND. Diseased leaves were incubated to promote sporulation. Ten single-spore isolates of P. teres collected from each location each year were tested for virulence by inoculation on 20 commonly used barley net blotch differential lines. Among the 100 isolates collected, one isolate collected in Fargo in 2006 (FGOH06Pt-8) and one isolate collected in Langdon in 2008 (LDNH08Pt-4) were identified as P. teres f. maculata due to their induction of spot-type lesions across the differential set. Conidial morphology of the two isolates was similar to P. teres f. teres isolates. A pathogenicity test of all isolates was performed on regional barley cvs. Tradition, Robust, and Lacey as well as barley lines Rika and Kombar (1) as previously described (3). The net form isolate 0-1 and spot form isolate DEN2.6 (obtained from B. Steffenson, University of Minnesota) were used as controls. The P. teres f. teres isolate 0-1 produced typical net type symptoms on all barley lines except the resistant line Rika, in which only small, dark spots were observed. DEN2.6 produced pin-point spot-like lesions with an extensive yellow halo on Robust, Lacey, Rika, and Kombar, but without chlorosis on Tradition. The two newly identified isolates induced elliptical spot-type lesions measuring 3 × 6 mm, larger than those produced by P. teres f. maculata isolate DEN 2.6, suggesting a higher level of virulence. We constructed a neighbor-joining phylogenetic tree using ClustalW2 ( http://www.ebi.ac.uk/ ) based on sequence identity of the internal transcribed spacer (ITS) region from 0-1 (GenBank No. GU014819), DEN2.6 (GenBank No. GU014820), FGOH06Pt-8 (GenBank No. GU014821), and LDNH08Pt-4 (GenBank No. GU014822) as well as P. teres f. maculata, P. teres f. teres, and P. tritici-repentis (causal agent of tan spot of wheat) accessions obtained from GenBank (2). All P. teres isolates clustered together and were clearly separated from the P. tritici-repentis cluster. Isolates FGOH06Pt-8 and LDNH08Pt-4 had identical ITS sequences and differed from DEN2.6 by only a single nucleotide. To our knowledge, this is the first report of P. teres f. maculata in North Dakota. Resistance to SFNB should now be considered in local barley breeding programs and cultivar releases. Reference: (1) M. Abu Qamar. Theor. Appl. Genet. 117:1261, 2008. (2) R. M. Andrie et al. Fungal Genet. Biol. 45:363, 2008. (3) Z. Lai et al. Fungal Genet. Biol. 44:323, 2007. (4) M. S. McLean et al. Crop Pasture Sci. 60:303, 2009.

Spot form of net blotch (SFNB) ( Pyrenophora teres f. maculata ) is an economically damaging foliar disease of barley in many of the world's cereal-growing areas. The gene Rpt4 that confers seedling resistance to SFNB has been mapped on the long arm of chromosome 7H, but no genes for adult plant resistance (APR) have been identified. A lack of field resistance to SFNB in breeders' lines selected for Rpt4 led us to investigate the genetics of APR to this disease. Five doubled-haploid populations were phenotyped for seedling and adult plant reaction. Markers linked to Rpt4 explained a large part of the seedling variation, but little of the APR. In 2 mapped populations, major quantitative trait loci (QTLs) for APR distal to Rpt4 on chromosome 7H were identified. QTLs contributing to APR on chromosomes 4H or 5H were also identified in each population. Association of the 5H QTL with a gene for cereal cyst nematode resistance and the probable effect of this linkage on the historical development of cultivars with adult plant resistance to SFNB is discussed.

Evaluation of resistance to Pyrenophora teres f. maculata in barley breeding populations via association mapping revealed a complex genetic architecture comprising a mixture of major and minor effect genes. In the search for stable resistance to spot form of net blotch (Pyrenophora teres f. maculata, SFNB), association mapping was conducted on four independent barley (Hordeum vulgare L.) breeding populations comprising a total of 898 unique elite breeding lines from the Northern Region Barley Breeding Program in Australia for discovery of quantitative trait loci (QTL) influencing resistance at seedling and adult plant growth stages. A total of 29 significant QTL were validated across multiple breeding populations, with 22 conferring resistance at both seedling and adult plant growth stages. The remaining 7 QTL conferred resistance at either seedling (2 QTL) or adult plant (5 QTL) growth stages only. These 29 QTL represented 24 unique genomic regions, of which five were found to co-locate with previously identified QTL for SFNB. The results indicated that SFNB resistance is controlled by a large number of QTL varying in effect size with large effects QTL on chromosome 7H. A large proportion of the QTL acted in the same direction for both seedling and adult responses, suggesting that phenotypic selection for SFNB resistance performed at either growth stage could achieve adequate levels of resistance. However, the accumulation of specific resistance alleles on several chromosomes must be considered in molecular breeding selection strategies.

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