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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]