Net blotch management in barley: Net form or spot form – does it matter?

During 2018, we observed an unusual morphology of net blotch strains growing on agar plates, compared with strains isolated from other samples and in previous years. Using DNA-based molecular tools, we established that these isolates represented the spot form of net blotch (Pyrenophora teres f. maculata (Ptm)). This form differs from the net form of net blotch (P. teres f. teres (Ptt)) regarding symptom development. They are considered genetically distinct species. The use of healthy seeds and resistant cultivars can help manage this barley disease, although reliable control often requires seed treatments and foliar fungicides. At present, there is a lack of information about whether the net blotch form(s) present affect the optimum management strategy.

Objectives

The first objective aimed to determine whether the two net blotch fungi have different levels of fungicide sensitivity. Quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) and azole resistance have been reported for net blotch on barley. The second objective aimed to develop DNA PCR-based tools to detect, identify and quantify both species in leaves and in seeds. Visual assessment to distinguish both diseases is difficult, especially in mixed infections. However, it is important to understand which species is present, because cultivars can have different R-genes for both diseases.

Key results

Six barley leaf samples with blotches, from five sites in 2018 – Cornwall (cv Propino), Dorset (cultivar unknown), Hampshire (cv Propino), Norfolk (cv Flagon) and Yorkshire (cv Tower) – were examined in this study. Ptm was isolated from the early and late season sample from Yorkshire (cv Tower), whereas Ptt was isolated from all the other samples. Other fungi that were simultaneously isolated from net-blotch infected leaves included Alternaria infectoria and Microdochium phragmitis.

Using reference isolates to check for specificity, a multiplex PCR targeting fungal ITS and P. teres species-specific markers was developed to identify fungal isolates as Ptt, Ptm or as another fungal species. A second multiplex PCR targeting barley and the P. teres species-specific markers was used to screen archived barley grain samples (1852 to 2018) from the Hoosfield long-term spring barley experiment at Rothamsted for presence of Ptt and Ptm. Ptt was detected for the first time in 1890, with 21 positive reactions since this time. Ptm was only detected in four samples (1982, 1995, 2001 and 2012), with both species only once simultaneously present in 2012.

The Ptm population sampled in Yorkshire was more sensitive to azole and SDHI fungicides, relative to the Ptt populations sampled in four UK regions. Mutations leading to amino acid substitutions SdhD-G138V or SdhD-D145G were detected in SDHI-insensitive Ptm isolates, growing at 1.0 ppm of fluxapyroxad as highest concentration. Amino acid substitution SdhC-R64K was found in all Ptm isolates, including old reference strains which were never exposed to SDHI fungicides and is not likely to affect SDHI binding. The most sensitive Ptm strain did not carry any Sdh mutations known to affect SDHI sensitivity and was only able to grow at 0.1 ppm fluxapyroxad as highest concentration. Mutations SdhC-S135R and SdhD-H134R, known to affect SDHI binding, were detected in the majority of the SDHI-insensitive Ptt strains that were able to grow in the presence of 10 ppm of fluxapyroxad as highest concentration. The most SDHI-insensitive Ptt strain showed partial growth at 100 ppm fluxapyroxad and carried SdhC-S135R in combination with D-G138V. The majority of Ptm strains were able to grow at 1.0 ppm epoxiconazole, with a few showing partial growth at 10 or 100 ppm. The most sensitive Ptm isolate was only able to grow at 0.1ppm epoxiconazole as highest concentration. In contrast, the majority of Ptt strains showed some partial growth at 100 ppm of epoxiconazole, the highest concentration tested.

Conclusions and recommendation for further research

1. Net blotch disease diagnosis and epidemics

Archived grain samples show that the spot form is not common, compared with the net form. However, the spot form has recently become a problem of epidemic proportions in several important barley-producing regions worldwide. With the spot form detected in some UK samples, it could become more prevalent. Regarding improving net blotch disease diagnosis and the identification of factors that drive epidemics, we recommend the following research initiatives:

• Continued exploration of archived samples of barley leaves and grains (from the long-term Hoosfield experiment) to identify which factors drive net blotch epidemics
• Screening seed lots for presence of Ptm and Ptt when setting up variety trials, as part of efforts to assess host resistance
• Development of quantitative PCR assays, to quantify Ptm and Ptt disease levels on grains and leaves
• Development of rapid tests for detection of both forms of net blotch (and ramularia). Based on Loop Mediated Isothermal Amplification (LAMP), these can use species-specific DNA sequences. LAMP tests, typically carried out on location within 30 minutes, can assist training and help staff visually assess difficult-to-diagnose diseases in Recommended List (RL) trials, especially when mixed infections are present

2. Fungicide sensitivity

Mycelium plug-based fungicide sensitivity assays show Ptt is less sensitive to azole and SDHI fungicides, compared to Ptm. Due to irregular radial growth, results for Ptm were more variable. Highly SDHI-insensitive isolates of Ptt carried SdhC-S135R or SdhD-H134R, while the least sensitive isolate carried a combination of SdhC-S135R and SdhD-G138V. SDHI-insensitive Ptm isolates carried SdhD-G138V or SdhD-D145G. Regarding fungicide sensitivity monitoring and understanding fungicide sensitivity shifts, we recommend the following research initiatives:

• Using high-throughput microtitre plate-based tests of spores to improve the sensitivity and accuracy of fungicide sensitivity testing for Ptt and Ptm
• Monitoring of SDHI sensitivity shifts in Ptt and Ptm populations, especially following the emergence of novel SDHI-resistant genotypes
• Conducting comparisons of the sensitivities of old reference strains with current populations to establish if the azole sensitivity in UK populations of Ptt and Ptm has shifted
• Using molecular approaches to elucidate the azole resistance mechanisms in UK populations of Ptt and Ptm
• Improving prediction of fungicide resistance development and identification of novel fungicide resistant alleles, through spore trapping in combination with NGS of PCR amplicons targeting fungicide resistance markers