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Monitoring virulent strains within pathogen populations is crucial to improve host resistance deployment strategies. Such monitoring increasingly involves field pathogenomics studies of molecular polymorphisms in pathogen genomes based on high‐throughput screening technologies. However, it is not always straightforward to predict virulence phenotypes from these polymorphisms, and in planta phenotyping remains necessary. We developed a method for ‘bulk phenotyping on checkerboard microcanopies of wheat near‐isogenic lines’ (BPC) for estimating the frequency of virulence against a resistance gene in mixed populations of the fungal pathogen Zymoseptoria tritici, the causal agent of Septoria tritici blotch (STB) in wheat, without the need for strain‐by‐strain pathogen phenotyping. Our method involves the uniform inoculation of a microcanopy of two wheat lines—one with the target resistance gene and the other without it—with a multistrain mixture of isolates representative of the population to be characterized, followed by the differential quantification of infection points (lesions). Using Stb16q, a wheat resistance gene that has recently broken down in Europe, we found a robust correlation between the ratio of the mean number of lesions on each wheat line and the frequency of virulent strains in the inoculum. Using pairs of virulent and avirulent strains, as well as synthetic populations consisting of 10 virulent strains and 10 avirulent strains mixed in different proportions, we validated the principle of the method and established standard curves at virulence frequencies close to those observed in natural conditions. We discuss the potential of this method for virulence monitoring in combination with molecular methods. [ABSTRACT FROM AUTHOR]

Zymoseptoria tritici is the causal fungus of Septoria tritici blotch (STB), a globally important foliar disease of wheat. Infections by more than one strain of a pathogen, or co‐infections, are known to be the most prevalent form of infection in the field. The impact that co‐infection of Z. tritici has on disease severity for the plant host and its effect on plant defence are not fully understood. Here, isolates of Z. tritici were extracted from STB‐presenting leaves from UK sites. Disease assays comparing single‐isolate inocula to mixed inocula were carried out on three cultivars of wheat, Flame, Stigg and Longbow. Leaves infected with mixed inocula were found to show low disease symptoms, including lesion and pycnidial coverage, compared with those infected with single isolates. Growing mixed isolates together in liquid media did not reveal evidence of competition or decreased growth compared with single‐isolate inocula. Wheat infected with a mixture of at least two Z. tritici isolates (of differing aggressiveness) could induce Pr‐1 (Pathogenesis‐related 1) at a significantly higher level in distal uninfected leaves compared with distal leaves of plants infected with a single aggressive isolate. Expression of Pr‐1 in plants with mixed inocula was intermediate to plants infected with an aggressive isolate (lower expression) and plants infected with a less aggressive isolate (higher expression). This suggests that wheat undergoes systemic "sensitization" by the least aggressive Z. tritici isolate during co‐infection. [ABSTRACT FROM AUTHOR]

Research to identify appropriate biological agents for controlling pathogens might exploit experimental evolution to select for enhanced antagonism against pathogens. We developed an experimental regime facilitating this approach, featuring the bacterial broad‐spectrum predator Myxococcus xanthus and the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that Z. tritici, a major cause of crop loss and fungicide use worldwide, both fuels growth of and is killed by M. xanthus on wheat straw, a probable source of seedling infection. M. xanthus was found capable of growth on moistened straw alone, unaided by Z. tritici, in a manner dependent on both M. xanthus initial density and on how long straw was moistened before M. xanthus inoculation. Such growth of non‐cellulolytic myxobacteria on plant detritus may have implications for understanding their roles in nutrient cycling. However, straw with Z. tritici was found to fuel greater M. xanthus growth than straw alone under many conditions. After shorter moistening periods, growth by low‐density M. xanthus populations was found to be fully dependent on Z. tritici. Such pathogen‐dependent M. xanthus populations could be sustained with population replacement over five 1‐week growth cycles, indicating the feasibility of conducting long‐term experimental evolution with this system. Furthermore, M. xanthus was found to kill majorities of Z. tritici populations on both buffered agar and straw. Our results suggest that myxobacteria may serve as effective biocontrol agents of Z. tritici and are amenable to long‐term experimental selection for enhanced killing of this pathogen, an approach broadly applicable to many potential biocontrol agents and target pathogens. [ABSTRACT FROM AUTHOR]

Septoria tritici blotch (STB) is one of the main causes of wheat yield loss in the world. Apart from good agrotechnical practice, disease‐resistant cultivars are required to prevent yield losses. Breeding of such cultivars is time‐consuming and laborious, mainly due to the quantitative character of such resistance, but can be shortened using precise phenotyping. We show that fluorescence parameters, collected in a high‐throughput system, can be used for estimating wheat STB resistance. Using machine learning methods (deep learning network, random forest), we demonstrate that disease resistance based on the percentage of necrotic leaf area can be estimated by alterations in fluorescence parameters 8 days after inoculation, and before disease symptoms become visible. Moreover, we applied a random forest classifier to assess the significance of fluorescence parameters in an informative way (classification accuracy = 64.84%, p value [Accuracy >No information rate] <0.001). Based on this, we observed extensive alterations in parameters of sensitive cultivars, reflecting worse photosynthetic performance. The most highly altered parameters were fluorescence intensity measurements and descriptors of energy flux through photosystem II (PSII) reaction centres towards PSI such as F2, F3 and F4 fluorescence intensities at 0.1, 0.270 and 2 ms; initial to maximal fluorescence ratio (F0/Fm); the time derivative of relative variable fluorescence (dV/dT0); maximum quantum yield of PSII in the dark‐adapted state (Fv/Fm); maximum electron transport flux at PSII (ET0/RC); maximum trapped excitonic energy flux per excited PSII cross section (CS) at time T0 (TR0/CS0); and quantum efficiency of the reduction of end acceptors dR0 [dR0/(1 − dR0)]. [ABSTRACT FROM AUTHOR]

Globally, bread wheat production is threatened by fungal diseases, including the devastating disease Septoria tritici blotch (STB). Given the global importance of STB, and the difficulty in identifying novel sources of resistance to this disease, we screened a variety of wheat genotypes, including wild, ancestral, and mutagenized lines, for their STB response. This delineated a panel of wild wheat relatives and Watkins collection lines with exceptional resistance to a range of Zymoseptoria tritici isolates, some of which are highly virulent on modern, elite wheat varieties. Additionally, we characterized the STB susceptibility of 500 lines of the wheat cultivar Cadenza TILLING population and developed backcross derivatives of two TILLING lines that show dominant partial resistance to STB. These backcross lines are partially resistant to multiple isolates of Z. tritici, and, with the wild and ancestral lines identified, provide a useful reservoir of STB‐resistant germplasm for use in wheat breeding programmes. [ABSTRACT FROM AUTHOR]

Demethylation inhibitor (DMI) and succinate dehydrogenase inhibitor (SDHI) fungicides are currently relied upon for the control of septoria tritici blotch (STB) in European wheat fields. However, multiple mutations have occurred over time in the genes encoding the targeted proteins that have led to a practical loss of fungicide efficacies. Among the different amino acid substitutions in Zymoseptoria tritici associated with resistance to these fungicides, S524T in CYP51 (DMI target) and H152R in SdhC (SDHI target) are regarded as conferring the highest resistance factors to DMI and SDHI, respectively. To facilitate further studies on the monitoring and selection of these substitutions in Z. tritici populations, a multiplex allele‐specific quantitative PCR (qPCR) assay allowing for estimation of both allele frequencies in bulk DNA matrices was developed. The assay was then used on complex DNA samples originating from a spore trap network set up in Belgium, Denmark, Sweden, and Ireland in 2017 and 2018, as well as on leaf samples with symptoms. The S524T allele was present in all field samples and its proportion was significantly higher in Ireland than in Belgium, whereas the proportion of H152R was only sporadically present in both countries. The frequency of S524T varied greatly in the airborne inoculum of all four countries; however, the H152R allele was never detected in the airborne inoculum. The method developed in this study can be readily adopted by other laboratories and used for multiple applications including resistance monitoring in field populations of Z. tritici. [ABSTRACT FROM AUTHOR]

Septoria tritici blotch (STB) disease of wheat is caused by the fungal pathogen Zymoseptoria tritici. It is the most important foliar disease of wheat in western Europe and affects wheat cultivation worldwide. The combination of intensive fungicide usage, a polycyclic asexual life cycle and an active sexual cycle has led to the emergence of fungal strains resistant/tolerant to all the major classes of fungicides used in its control. The hallmark of this disease is a long, symptomless latent phase that precedes the onset of visible symptoms. Understanding the processes that occur during the symptomless phase of infection is paramount in developing alternative strategies for disease control; however, large gaps in our knowledge of the disease remain. The known unknowns of the latent stage of infection can be summarized in three questions. Does the fungus initiate or manipulate host defences to trigger programmed cell death in order to facilitate nutrient acquisition or is the host acting exclusively? Does the fungus feed during both the latent phase and the necrotrophic phase like a true hemibiotroph? Does the long latent phase serve a beneficial function for the fungus or is it simply an artefact of evolution? This review aims to distil observations made during studies that have directly or indirectly contributed to answering these questions and points towards their most likely answers. [ABSTRACT FROM AUTHOR]

Zymoseptoria tritici, the causal agent of septoria tritici blotch (STB), remains a significant threat to European wheat production with the continuous emergence of fungicide resistance in Z. tritici strains eroding the economic sustainability of wheat production systems. The life cycle of Z. tritici is characterized by a presymptomatic phase (latent period, LP) after which the pathogen switches to an aggressive necrotrophic stage, when lesions bearing pycnidia quickly manifest on the leaf. As minimal knowledge of the possible role of the LP in supporting STB resistance/susceptibility exists, the goal of this study was to investigate the spatial and temporal association between the LP and disease progression across three locations (Ireland – Waterford, Carlow; UK – Norwich) that represent commercially high, medium and low STB pressure environments. Completed over two seasons (2013–2015) with commercially grown cultivars, the potential of the LP in stalling STB epidemics was significant as identified with cv. Stigg, whose high level of partial resistance was characterized by a lengthened LP (c. 36 days) under the high disease pressure environment of Waterford. However, once the LP concluded it was followed by a rate of disease progression in cv. Stigg that was comparable to that observed in the more susceptible commercial varieties. Complementary analysis, via logistic modelling of intensive disease assessments made at Carlow and Waterford in 2015, further highlighted the value of a lengthened LP in supporting strong partial resistance against STB disease of wheat. [ABSTRACT FROM AUTHOR]

The use of cultivar mixtures to control foliar fungal diseases is well documented for windborne diseases, but remains controversial for splash-dispersed diseases. To try to improve this strategy, a cultivar mixture was designed consisting of two wheat cultivars with contrasted resistance to Mycosphaerella graminicola, responsible for the rainborne disease septoria tritici blotch ( STB), in a 1:3 susceptible:resistant ratio rather than the 1:1 ratio commonly used in previous studies. The impact of natural STB epidemics in this cultivar mixture was studied in field experiments over 4 years. Weekly assessments of the number of sporulating lesions, pycnidial leaf area and green leaf area were carried out on the susceptible cultivar. In years with sufficient STB pressure, disease impacts on the susceptible cultivar in the mixture were always significantly lower than in the pure stand (e.g. 42% reduction of pycnidial leaf area for the three upper leaves in 2008 and 41% in 2009). In years with low STB pressure (2010 and 2011), a reduction of disease impacts was also shown but was not always significant. After major rainfall events, the number of sporulating lesions observed on the susceptible cultivar after one latent period was reduced on average by 45% in the mixture compared to the pure stand. All the measurements showed that a susceptible cultivar was consistently protected, at least moderately, in a mixture under low to moderate STB pressure. Therefore, the results prove that the design of an efficient cultivar mixture can include the control of STB, among other foliar diseases. [ABSTRACT FROM AUTHOR]

To provide insight into the genetic structure of Mycosphaerella graminicola populations in Iran, a total of 221 isolates were collected from naturally infected wheat fields of five major wheat-growing provinces and analysed using AFLP markers and mating-type loci. All populations showed intermediate to high genotypic diversity. In the Golestan and Ardabil populations two mating types were found at near-equal frequencies, whilst all populations were in gametic disequilibrium. Moreover, clonal haplotypes were identified in different sampling sites within a field in both the Khuzestan and Fars provinces, demonstrating that pycnidia are probably the primary source of inoculum. All five populations had low levels of gene diversity and had private bands. Low levels of gene flow and high genetic differentiation were observed among populations and different clustering methods revealed five genetically distinct groups in accordance with the sampling areas. The Golestan and East Azarbaijan populations were more genetically differentiated than the others. Random genetic drift, selection and geographic barriers may account for the differentiation of the populations. The results of this study indicate a population structure of M. graminicola in Iran contrasting to that of most other countries studied. [ABSTRACT FROM AUTHOR]

The association of the cell wall degrading enzyme endo-beta-1,4-xylanase (EC 3.2.1.8) with pathogenicity of Mycosphaerella graminicola was examined in planta. The enzyme production of two M. graminicola isolates (T0372 and T0491), as well as their ability to infect seedlings of susceptible wheat cv. Scorpion, was first compared. No significant difference was found between the two isolates regarding spore germination rates, mycelial growth on the leaf surface or direct and stomatal penetrations. However, restricted hyphal growth was observed inside leaves inoculated with T0372, whereas successful mesophyll colonization with a strong intercellular fungal growth was found in leaves infected with T0491. Likewise, T0372 was unable to induce lesions bearing pycnidia and to produce endo-beta-1,4-xylanase activity until 22 days post-inoculation (d.p.i.). On the other hand, significant high increases of both diseased leaf area bearing pycnidia and endo-beta-1,4-xylanase activity were observed between 16 and 22 d.p.i. for T0491 ( r = 0·98). The investigation of 24 additional isolates, including the IPO323 and IPO94269 reference isolates, highlighted a strong correlation between endo-beta-1,4-xylanase activity and disease development levels ( r = 0·94). This study demonstrates that differences in pathogenicity in M. graminicola are not linked to events on the leaf surface or to frequency of leaf penetration, but to the ability of the fungus to colonize the mesophyll and to produce the cell wall degrading enzyme endo-1,4-beta-xylanase during the necrotrophic phase. [ABSTRACT FROM AUTHOR]

This study reports the discovery of a gene for resistance to septoria tritici blotch (STB) in two spring wheat cultivars, Courtot and Tonic. The gene, named Stb9, confers resistance to Mycosphaerella graminicola isolate IPO89011. It was mapped by quantitative trait loci (QTL) analysis using an existing map of Courtot × Chinese Spring and was located between markers Xfbb226 (3·6 cM) and XksuF1b (9 cM) on the long arm of chromosome 2B. Markers linked to Stb9 in Courtot were then shown to be linked to resistance to IPO89011 in F3 families of Tonic × Longbow. Allelism tests in which Tonic was crossed with Courtot confirmed that Tonic has a gene for resistance to IPO89011 at or very close to the Stb9 locus. SSR markers flanking Stb9 may be used in marker-assisted selection to introgress this gene into winter cultivars or in spring wheat breeding programmes outside Europe. [ABSTRACT FROM AUTHOR]

Genes for specific resistance to European and American isolates of Mycosphaerella graminicola, the causal agent of septoria tritici blotch (STB) of wheat, have been identified and mapped in various cultivars and breeding lines and are distributed throughout the genome. The location of a gene for resistance to an Ethiopian isolate, IPO88004, which is currently the most widespread resistance present in European wheat cultivars, is reported. The resistance was mapped in the Swiss cultivar Arina which, besides high partial resistance to STB, also has specific resistance to IPO323, controlled by Stb6 and to IPO88004. An F5 recombinant inbred population from a cross between Arina and the susceptible cultivar Forno was tested in whole seedling trials. Using multiple QTL mapping (MQM), a gene for resistance to M. graminicola isolate IPO88004 in cv. Arina was located to chromosome 6AS. The gene is named Stb15. Seedling tests on a double haploid population of cvs Arina × Riband indicated that the UK wheat cv. Riband also has Stb15 or another gene for specific resistance to IPO88004 allelic or closely linked to Stb15. [ABSTRACT FROM AUTHOR]

From a total of 238 European cultivars and breeding lines screened for isolate-specific resistance to septoria tritici blotch (STB) with eight Mycosphaerella graminicola isolates from five different countries, 142 lines were resistant to Ethiopian isolate IPO88004, and 43 lines were specifically resistant to IPO323, with little or no leaf area bearing pycnidia of M. graminicola. These lines probably all have the resistance gene Stb6. Specific resistances to isolates CA30JI, IPO001, IPO89011, IPO92006 and ISR398 were less common. Seventy-three per cent of the lines were specifically resistant to at least one isolate and 36 lines were resistant to more than one isolate. The line with the greatest number of specific resistances was the spring cultivar Raffles, with five. The most resistant line in which no specific resistance was identified was the Italian landrace Rieti, an ancestor of many modern European wheat cultivars. There was also a wide range of partial resistance among the lines tested, expressed in detached seedling leaves. Information about the resistance of wheat lines to M. graminicola isolates will assist breeders to choose parents of crosses from which progeny with superior resistance to STB may be selected. [ABSTRACT FROM AUTHOR]

Septoria tritici blotch (STB) is a major disease of wheat, reaching epidemic proportions in many parts of the world. In several studies, taller, later-maturing cultivars have had lower disease levels. This study was undertaken to investigate the genetic associations of natural field infection by STB with disease-escape mechanisms related to heading date and height components, mainly leaf spacing, in a population where height and maturity are not controlled by major genes. In field trials of a single seed-descent population of a cross between two nonsemi-dwarf cultivars, Apollo (with strong partial resistance to STB) and Thésée (susceptible), conducted over 3 years, there was a negative correlation between STB and heading date. There was no correlation between STB and distance from stem base to leaf 2; and there was an unexpected positive correlation between STB and distance from flag leaf to leaf 2, which contradicted the so-called ‘ladder effect’ postulated in STB epidemiology. No effect was detected of the presence of the 1BL−1RS translocation on STB levels. The largest single contributor to variation in STB levels was genetic variation between the progeny lines, and the narrow-sense heritability was 42%. These results suggest that breeders can select for STB resistance alongside optimal stature within the range of height which is adaptive in a particular environment. [ABSTRACT FROM AUTHOR]

Fifty-two wheat cultivars and breeding lines, most of which have been used in breeding programmes worldwide, were tested for isolate-specific resistance toMycosphaerella graminicolaisolate IPO323, which interacts with theStb6gene of wheat (first identified in cvs Flame and Hereward) via a gene-for-gene relationship. Twenty-three lines were specifically resistant to this isolate. Sixteen resistant lines were crossed with Flame for a test of allelism. All progeny lines were resistant, suggesting that the 16 parental lines hadStb6, a gene allelic to it or a gene closely linked to it. In 14 lines, resistance to IPO323 was controlled byStb6only. An exception was Kavkaz-K4500 L6.A.4., which has two genes for resistance to IPO323, one of which isStb6. The microsatellite markerXgwm369was used to examine genetic diversity in the region of the genome containingStb6, to which it is closely linked. Eleven alleles ofXgwm369, with amplified fragments of 10 different sizes, as well as apparent nonamplification of this marker in Bulgaria 88, were detected. Through the use of information about lines’ ancestry, combined withXgwm369alleles, it was shown thatStb6entered world wheat-breeding programmes on a minimum of six occasions, and possibly from as many as 11 sources. The presence ofStb6in both European and Chinese landraces suggests that this gene has been present in cultivated wheat since the earliest times of agriculture. [ABSTRACT FROM AUTHOR]

Twenty-four wheat cultivars and breeding lines were screened for isolate-specific resistance to septoria tritici blotch (STB) caused by 12 isolates of Mycosphaerella graminicola. New isolate-specific resistances that could be used in wheat breeding were identified. Major sources of resistance to STB used in world breeding programmes for decades, such as Kavkaz-K4500, Veranopolis, Catbird and TE9111, have several isolate-specific resistances. This suggests that ‘pyramiding’ several resistance genes in one cultivar may be an effective and durable strategy for breeding for resistance to STB in wheat. Several cultivars, including Arina, Milan and Senat, had high levels of partial resistance to most isolates tested as well as isolate-specific resistances. Resistance to isolate IPO323 was common, present in all but one of the major sources of resistance tested. This suggests that resistance to IPO323 may be an indicator of varietal resistance to STB in the field. [ABSTRACT FROM AUTHOR]

Isolate-specific resistance of 71 cultivars and breeding lines of wheat (Triticum aestivum) to septoria tritici blotch was evaluated in six field trials in the Netherlands, Switzerland and the UK between 1995 and 1997. Each plot was inoculated with one of six single-pycnidium isolates of the pathogen Mycosphaerella graminicola. There were strong interactions between wheat lines and M. graminicola and the line-by-isolate interactions were stable over the six trials. Lines with specific resistance or specific susceptibility to each of the isolates were identified. Specific resistance to isolate IPO323 was especially common, being carried by 22 lines from 10 countries. The results confirm that line-by-isolate interactions in septoria tritici blotch of wheat are effective in adult plants in field conditions, and are not simply confined to seedlings. Wheat lines with good, quantitative resistance to all or most isolates were identified, including lines from Brazil, the USA and seven European countries. These may be useful as sources of resistance in wheat breeding. [ABSTRACT FROM AUTHOR]

A detached seedling leaf technique was developed to screen for resistance to septoria tritici blotch of wheat and to detect specific interactions between cultivars and isolates. Wheat seedlings were inoculated with spore suspensions of Mycosphaerella graminicola. Detached primary leaves were then placed in a clear plastic box such that their cut ends were sandwiched between layers of agar containing benzimidazole, with a gap below the middle of the leaves. Mean levels of disease were affected by light and temperature, and also by the concentration of benzimidazole, such that higher concentrations resulted in less disease. Second leaves were more susceptible than seedling primary leaves. However, none of these factors affected ranking of disease among cultivars or cultivar-by-isolate interactions. Kavkaz–K4500 1.6.a.4, Synthetic 6x and Triticum macha showed specific susceptibility and resistance to different isolates. The detached leaf technique could be a useful complement to field trials and an alternative to whole seedling assays in assessing cultivar resistance and investigating the genetics of the host–pathogen interaction. [ABSTRACT FROM AUTHOR]