Barley Spot Research Articles

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.

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.

Morphological Signs of Barley Spot Pathogens in the Conditions of the Almaty Region

Morphological Signs of Barley Spot Pathogens in the Conditions of the Almaty Region

Genome Wide Association Mapping of Spot Blotch Resistance at Seedling and Adult Plant Stages in Barley.

Barley spot blotch (SB) caused by Cochliobolus sativus is one of the major constrains to barley production in warmer regions worldwide. The study was undertaken to identify and estimate effects of loci underlying quantitative resistance to SB at the seedling and adult plant stages. A panel of 261 high input (HI-AM) barley genotypes consisting of released cultivars, advanced breeding lines, and landraces, was screened for resistance to SB. The seedling resistance screening was conducted using two virulent isolates from Morocco (ICSB3 and SB54) while the adult plant stage resistance was evaluated at two hot spot locations, Faizabad and Varanasi, in India under artificial inoculation using a mixture of prevalent virulent isolates. The HI-AM panel was genotyped using DArT-Seq high-throughput genotyping platform. Genome wide association mapping (GWAM) was conducted using 13,182 PAV and 6,311 SNP markers, for seedling and adult plant resistance. Both GLM and MLM model were employed in TASSEL (v 5.0) using principal component analysis and Kinship Matrix as covariates. Final disease rating and Area Under Disease Progress Curve (AUDPC) were used for the evaluation of adult stage plant resistance. The GWAM analysis indicated 23 QTL at the seedling stage (14 for isolate ICSB3 and 9 for isolate SB54), while 15 QTL were detected at the adult plant stage resistance (6 at Faizabad and 9 at Varanasi) and 5 for AUDPC based resistance at Varanasi. Common QTL at seedling and adult plant stages were found across all barley chromosomes. Seedling stage QTL explained together 73.24% of the variance for seedling resistance to isolate ICSB3 and 49.26% for isolate SB54, whereas, QTL for adult plant stage resistance explained together 38.32%, 44.09% and 26.42% of the variance at Faizabad and Varanasi and AUDPC at Varanasi, respectively. Several QTL identified in this study were also reported in previous studies using bi-parental and association mapping populations, corroborating our results. The promising QTL detected at both stages, once validated, can be used for marker assisted selection (MAS) in SB resistance barley breeding program.

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.

СЕЛЕКЦИЯ НА ПОВЫШЕНИЕ АДАПТИВНОГО ПОТЕНЦИАЛА ЯРОВОГО ЯЧМЕНЯ ПО УСТОЙЧИВОСТИ К ЛИСТОВЫМ ПЯТНИСТОСТЯМ В ЦЕНТРАЛЬНОМ РЕГИОНЕ РОССИИ

To solve the problem of long-term barley resistance to main biotic factors, the major role belongs to study of the original selection material and scientifically-based methods of parent choice. The goal of our study was to find the sources, characterized with high resistance to gelmintosporiosis pathogens of barley. The field environmental tests were laid up on the experimental fields of Moscow and Ryazan agricultural universities, in 2016-2018. In this article, the results of the spring barley sample collections studies, aimed toward their evaluation by the resistance to barley mesh barley spot and dark brown barley spot are presented. The immunology evaluation of the original material of different origin demonstrated, that the degree of gelmintosporiosis-caused barley diseases at experimental locations depended greatly of meteorological conditions per year. It was determined, that the greatest variation of barley spot resistance (V=46,8-69,8) was observed during the years of their maximum spread. The most valuable original material for cultivar’s adaptive potential increase are those genotypes, that have group resistance to diseases. Due to that, the main goal of our study was to evaluate the collection samples by their resistance to the local pathogens populations, and highlight the samples, simultaneously resistant to mesh barley spot and dark brown barley spot. Twelve samples were chosen, that demonstrated highest group resistance to gelmintosporiosis-caused diseases. By using the methods of stability and plasticity evaluation, we were able to determine the varietal differences in perspective material, and obtain the additional data to chose the most highly-adaptive forms. The results allowed us to recommend Nadejny, Talovsky 9, Binom, Krasnoyarujsky 6, Yakub, Getiman and Vziretz cultivars as initial material for new (with high and stable pathogen resistance) cultivars development. The evaluation of differentiating ability allowed to determine the selection environments with the best-developed pathogen complex as the best selection backgrounds for the selection of genotypes, characterized by high adaptive potential, stably maintaining the resistance gelmintosporiosis-caused barley spot regardless of weather conditions.

Expression Analysis of Pyrenophora teres f. maculata-Responsive Loci in Hordeum vulgare

Abstract Pyrenophora teres f. maculata is the causal agent of barley spot form net blotch (SFNB), a major stubble-borne disease in many barley-growing areas worldwide. In plants, the Nucleotide-Binding Site-Leucine-Rich Repeat (NBS-LRR) gene family functions in immunity against a variety of pathogens and pests. From a pre-established set of NBS-type resistance gene candidates, we have selected three candidate genes, HvNBS10, HvNBS72 and HvNBS85, to analyze their possible involvement in P. teres f. maculata resistance. The studied genes were mapped on chromosomes 5H and 7H. Expression profiles using qRT-PCR, 48 hours after infection by P. teres. f. maculata, revealed that the transcription of all genes acted in the same direction (down-regulation) in both resistant and susceptible cultivars, although they showed a variation in transcript dosage. This result suggests that coordinated transcriptional responses of multiple barley NBS genes would be required to an efficient response against P. teres f. maculata. Moreover, the phylogenetic analysis revealed that the studied barley candidate R genes were characterized by a high homology with the barley Nbs2-Rdg2a gene conferring resistance to the fungus Pyrenophora graminea, suggesting a common origin of P. graminea and P. teres resistance genes in barley, following pathogens evolution. The genes characterized in the present study hold potential in elucidating the molecular pathways and developing novel markers associated with SFNB resistance in barley.

Efficient Discovery of Single-Nucleotide Variations in Cochliobolus sativus Vegetative Compatibility Groups by Ecotilling

Cochliobolus sativus, the causal agent of barley spot blotch disease, is able to undergo spontaneous, vegetative cell fusion. In this study, the utility of enzymatic mismatch cleavage for discovery of single-nucleotide variations in Syrian Vegetative Compatibility Groups (VCGs) obtained from pairing complementary nit mutants of C. sativus isolates was investigated for the first time. Gene-specific primers were designed from the whole fungal genome for use in the Ecotilling assays on VCGs isolates. Differences of band patterns among the digested products of different isolates were clearly observed by agarose gel electrophoresis. Interestingly, isolates belonging to the same VCG had some different sets of single nucleotide polymorphisms. Twenty-six putative nucleotide polymorphisms were identified among the C. sativus VCGs. Therefore, the results suggested that this marker type may be informative for confirmation the conventionally determined VCGs by increasing the reliability of heterokaryons group classification.

Barley spot blotch intensity, damage, and control response to foliar fungicide application in southern Brazil

Barley spot blotch (SB) is a frequent foliar disease of the brewing barley crop in southern Brazil. At present, there are few reports regarding the efficacy of fungicide spray programs in relation to the severity of SB damage to barley crop yield. The goals of this study were as follows: 1) to determine the crop growth stage at which severity of SB was most strongly related to grain yield (GY), thousand kernel weight (TKW), and granulometry (G) damage in brewing barley and 2) to evaluate the efficacy of fungicides in controlling SB and the effect of fungicide application timing on disease intensity and yield. The experiments were performed using the BRS Cauê barley cultivar at Muitos Capões municipality, Rio Grande do Sul State, southern Brazil, during the 2009 and 2010 growing seasons. The experiments involved a randomized complete block design with five treatments and four replicates. The treatments varied by the number of fungicide applications. The control received no fungicide application. SB severity assessments were performed at the following growth stages: main stem and one tiller developed, main stem and four tillers developed, swelling 2nd node detectable, flag leaf ligule just visible, full boot, and 50% of spikes visible. SB severity data were integrated as a function of time to determine the area under the disease progress curve (AUDPC). SB resulted in damage of up to 49.2% and 34.6% in GY, 19.9% and 19.7% in TKW, and 26.0% and 11.2% in G in the 2009 and 2010 growing seasons, respectively. A frequency of three and four foliar fungicide applications controlled 70.1% and 78.3% of SB in the 2009 growing season and 85.8% and 88.6% in the 2010 growing season, respectively. GY and SB severity showed a significant negative relationship in both the 2009 and 2010 growing seasons and at all growth stages evaluated. The greatest GY damage related to SB severity occurred at the final growth stages at full boot and 50% of spikes visible. There was a significant negative relationship between AUDPC and GY, with damage coefficients of 17.19 and 22.94 in the 2009 and 2010 growing seasons, respectively. There was a positive net return on barley GY, TKW, and G when four fungicide applications were used for SB control.

Differential effects of spot blotch on photosynthesis and grain yield in two barley cultivars

Barley spot blotch (SB), caused by Cochliobolus sativus, is an important barley disease which causes extensive grain yield losses. These losses may not always correlate directly with the amount of diseased leaf area. Two barley cultivars, Quebracho (susceptible to SB) and Carumbe (with intermediate susceptibility to SB), were compared in field experiments in 2003, 2004 and 2006. Plots of each cultivar were either inoculated with C. sativus or protected with fungicide under field conditions to generate contrasting treatments: i) diseased, and ii) free of disease, respectively. SB severity over the growing season, photosynthetic rate on leaves with no visible symptoms and grain yield were assessed for each treatment and year. There was no treatment effect on cv. Carumbe, while cv. Quebracho showed a significant yield reduction, even though SB severity during the grain filling period was <10 %. This yield reduction was associated with a reduced photosynthetic rate at the beginning of the grain filling period in cv. Quebracho. A similar experiment was conducted under greenhouse conditions, adding a treatment without inoculum or fungicide. There were no differences in photosynthetic rate or grain yield per plant among treatments. These results suggest a distinct physiological response to SB infection among cultivars affecting leaf photosynthetic rate, and SB severity may not be the best estimator of yield losses caused by SB.

HYDROGEN PEROXIDE INDUCTION IN BARLEY- COCHLIOBOLUS SATIVUS INTERACTION

The fungus Cochliobolus sativus is the causal agent of Barley spot blotch (SB) disease. In order to better un- derstand barley physiological reaction towards this dis- ease, hydrogen peroxide (H2O2) and ion fluxes in two different genotypes inoculated with a virulent isolate of C. sativus were investigated. Results showed that SB de- velopment was directly dependent on H2O2 generation and accumulation during infection. Significant differ- ences (P<0.001) in H2O2 values were found among dif- ferent time points, with values being consistently higher in the resistant genotype. The interaction between host leaves and pathogen produced a marked increase in electrolyte leakage from the susceptible genotype com- pared with the resistant one. The information obtained from this study highlights crucial points that can guide hypothesis-driven research to elucidate the molecular mechanisms involved in C. sativus-barley interaction

HETEROGENEITY IN THE INTERNAL TRANSCRIBED SPACERS OF THE RIBOSOMAL DNA IN COCHLIOBOLUS SATIVUS AS REVEALED BY IRAP

Cochliobolus sativus, the causal agent of barley spot blotch disease, is an economically important fungus with a worldwide distribution. The heterogeneity within the nuclear rDNA [internal transcribed spacers (ITS)] of C. sativus isolates was studied using the amplified fragments through inter-retrotransposon amplified polymorphism (IRAP). A total of 173 scorable DNA bands were obtained from 7 primer pairs, and 20 of these (11.5%) were polymorphic. A neighbour-joining diagram, based on Nei’s genetic distances, showed that isolates formed three phylogenetic groups, and did not fall into clusters specific to geographical origin or colour of the isolate, which suggests a regional dispersal of C. sativus. The information obtained from this study is crucial for studying genetic variation in this fungus; in addition, IRAP-ITS markers can illustrate other types of polymorphism which can help to clarify the genetic structure of C. sativus isolates.

Effect of xanthan gum and allicin as elicitors against Bipolaris sorokiniana on barley in field experiments

Barley spot blotch, caused by the fungus Bipolaris sorokiniana (Sacc.) Shoem., leads to decreases in productivity and affects beer quality. The objective of the present study was to validate findings in laboratory and greenhouse experiments on biochemical responses that occur in malting barley cultivar BRS 195 when treated with elicitors of resistance (xanthan gum and allicin) and with the fungicide epoxiconazole + pyraclos- trobin, compared with untreated plants in field trials in 2003 and 2004. Experimental design was that of random blocks with four repetitions. The incidence and severity of the disease were evaluated, as well as the size of lesions and area infected. Remarkable was that the area infected on the leaves and the size of the lesions were smaller in the group treated only with the elicitors, whereas plants treated with the fungicide only had larger diseased areas. Plants in the control group presented different lesion sizes and the greatest infected area. The biochemical analyses were performed on: total protein concentration, activity of the enzyme s-1,3-glucanase and phenol concentration. Their variables were rather similar in both years of the trial. Total protein concentration and activity of s-1,3-glucanase was higher in the control plots than in plots treated with the two elicitors or with the fungicide. Phenol concentration was higher in the control and in the plot treated with the fungicide only. Allicin and xanthan gum used as elicitors were as effective as the fungicide against the leafspot severity on barley. Though lesions still were present on leaves the elicitors were as protective as the fungicide.

Vegetative compatibility and pathogenicity inCochliobolus sativus

Forty isolates of C. sativus, the causal agent of barley spot blotch, were isolated from symptomatic tissues of barley from different regions in Syria. These isolates were grouped into vegetative compatibility groups (VCGs) by demonstrating heterokaryosis by complementation tests using nitrate non-utilising (nit) mutants. All isolates were grouped into three VCGs, 1-A, 1-B and 1-C. No self-incompatibility was observed in any of the isolates tested. VCG 1-C was the most common group within growing regions in Syria and proved to be the most virulent of the VCGs identified. These data indicate that the level of virulence in C. sativus is related to VCG.

Molecular karyotyping and chromosome length polymorphism in Cochliobolus sativus

Fungi are known to have variable genomes that can generate new virulence types capable of attacking important crop plants. To assess chromosome length polymorphisms in the barley spot blotch pathogen ( Cochliobolus sativus), we analyzed the karyotypes of 16 isolates using contour-clamped homogeneous electric field (CHEF) electrophoresis. The collection of isolates studied were from diverse regions of the world (USA, Canada, Japan, Brazil, Uruguay, and Poland) and included representatives comprising the three known C. sativus pathotypes of 0, 1, and 2. Under two different running conditions, the number of CHEF bands observed ranged from 8 to 13 with a size range of 0.85 to 3.80 mega-bases (Mb). Each of the 16 isolates showed a unique banding pattern, except for two North Dakota isolates ND90Pr and ND91-Bowman, which were very similar. Single-copy DNA probes, previously assigned to each of the 15 chromosomes identified in reference isolate ND93-1, were hybridized to Southern blots of CHEF-separated chromosomes and revealed highly polymorphic chromosomes among isolates. Chromosomal rearrangements (translocations, deletions, duplications) were found in several isolates. DNA markers previously found linked to VHv1, a gene in pathotype 2 isolates conferring virulence on barley cultivar Bowman, also were used as probes in hybridizations with the CHEF blots. The results showed that the chromosome carrying the virulence gene in pathotype 2 isolates is larger than its counterpart without the gene in other isolates. This suggests that the genomic region carrying the virulence locus VHv1 is unique to pathotype 2 isolates. This study provides useful information on genome structure and divergence, which is essential for advancing our understanding of the genetics and biology of C. sativus.

Germinability of Cochliobolus sativus Conidia Exposed to Solar Radiation

AbstractUnder field conditions, conidia of Cochliobolus sativus, the causal agent of barley spot blotch, were exposed to direct solar radiation or placed in a ventilated enclosure in darkness for 0.5–12 h. In addition, conidia were either exposed or not exposed to UV light (254 nm) from 1 to 75 min in the laboratory. Conidia were placed after exposure on water agar in closed Petri dishes and allowed to germinate for 24 h. Germinability of conidia was reduced up to 99% after 12 h of exposure to solar irradiance (GS) (690–900 w/m2). Germinability of conidia that were not exposed to sunlight (GNS) did not decrease significantly during experiment time. Similar results were obtained when the variable (GNS−GS)/GNS was applied. Temperature accounted for limited variation in germinability, whereas solar radiation accounted for most of the variation. Germinability of conidia in the laboratory was reduced up to approximately 100% by doses of UV‐C of approximately 14.4 kJ/m2. Germinability of conidia in the field was reduced up to approximately 100% by doses of UV‐B of approximately 39.9 kJ/m2. The information obtained from this study will contribute to the development of a forecast system that incorporates the probability of incoming aerial spores.

PATHOTYPES OF COCHLIOBOLUS SATIVUS (SPOT BLOTCH) ON BARLEY IN SYRIA

In order to study the Syrian pathotype diversity of Cochliobolus sativus, the causal agent of barley spot blotch disease, a survey was conducted using 31 isolates and 13 barley genotypes. Four pathotypes (pt 1-4) were identified based on the lesion form and infection response of the genotypes with mean disease rating from 1.76 to 7.46. Pt 1 exhibited low virulence on all used genotypes, pts 2 and 3 were moderately virulent and pt 4 was highly virulent. The two most common pathotypes were pt 3 (35%) and pt 4 (42%). The barley genotype AECS 71 was highly resistant to all pathotypes suggesting the existence of a general resistance mechanism. This genotype may be recommended as a possible donor in breeding programmes. The information obtained from this study should facilitate deploying effective resistance to C. sativus in barley.