Non‐target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici

BACKGROUND

A new generation of more active succinate dehydrogenase (Sdh) inhibitors (SDHIs) is currently widely used to control Septoria leaf blotch in northwest Europe. Detailed studies were conducted on Zymoseptoria tritici field isolates with reduced sensitivity to fluopyram and isofetamid; SDHIs which have only just or not been introduced for cereal disease control, respectively.

RESULTS

Strong cross‐resistance between fluopyram and isofetamid, but not with other SDHIs, was confirmed through sensitivity tests using laboratory mutants and field isolates with and without Sdh mutations. The sensitivity profiles of most field isolates resistant to fluopyram and isofetamid were very similar to a lab mutant carrying SdhC‐A84V, but no alterations were found in SdhB, C and D. Inhibition of mitochondrial Sdh enzyme activity and control efficacy in planta for those isolates was severely impaired by fluopyram and isofetamid, but not by bixafen. Isolates with similar phenotypes were not only detected in northwest Europe but also in New Zealand before the widely use of SDHIs.

CONCLUSION

This is the first report of SDHI‐specific non‐target site resistance in Z. tritici. Monitoring studies show that this resistance mechanism is present and can be selected from standing genetic variation in field populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Multiplex qPCR assay for simultaneous quantification of CYP51-S524T and SdhC-H152R substitutions in European populations of Zymoseptoria tritici

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.     

Lack of cross-resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii

BACKGROUND: Recently in Japan, isolates resistant to boscalid, a succinate dehydrogenase inhibitor (SDHI), have been detected in Corynespora cassiicola (Burk. & Curt.) Wei and Podosphaera xanthii (Castaggne) Braun & Shishkoff, the pathogens causing Corynespora leaf spot and powdery mildew disease on cucumber, respectively. Resistant isolates of C. cassiicola are widely distributed and represent a serious problem in disease control at present. Novel SDHI fungicides, including fluopyram, are now under development. RESULTS: The growth of very highly boscalid‐resistant, highly resistant and sensitive isolates of C. cassiicola was strongly suppressed on fluopyram‐amended YBA agar medium. Although boscalid and another SDHI, penthiopyrad, hardly controlled Corynespora leaf spot and powdery mildew on cucumber plants when very highly or highly boscalid‐resistant isolates were employed for inoculation, fluopyram still exhibited excellent control efficacy against these resistant isolates as well as sensitive isolates of C. cassiicola and P. xanthii. CONCLUSION: Differential sensitivity to boscalid, penthiopyrad and fluopyram, clearly found in these two important pathogens of cucumber, may indicate involvement of a slightly distinct site of action for fluopyram from the two other SDHIs. This finding may lead to the discovery of unique SDHIs in the future. Copyright © 2011 Society of Chemical Industry     

Amino acid substitutions responsible for different QoI and SDHI sensitivity patterns in Puccinia horiana,the causal agent of chrysanthemum white rust

Quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) are major groups of agricultural fungicides. However, resistance to some of these fungicides has been reported in a Japanese population of Puccinia horiana, the causal agent of chrysanthemum white rust disease. Because their mechanisms are not well understood, we investigated the existence of mutations in QoI and SDHI target protein-encoding genes. Eight out of nine isolates from cultivated chrysanthemum carried L275F and L299F amino acid substitutions in cytochrome b, the target protein of QoIs. These isolates showed 23- and 17-fold higher EC₅₀ values for the QoI fungicides azoxystrobin and kresoxim-methyl, respectively, in basidiospore germination inhibitory tests, while they were hypersensitive to another QoI, famoxadone. All nine isolates were resistant to SDHI oxycarboxin and carried the I88F substitution in SdhC. This substitution was orthologous to the SdhC-I86F substitution found in some Brazilian isolates of the soybean rust fungus, Phakopsora pachyrhizi, showing reduced sensitivity to some SDHIs. Although the rarity of wild-type sensitive isolates, the subsequent limited number of comparisons between wild types and mutants, and a difficulty in applying reverse genetic analysis to this obligate parasite, are obstacles in making definitive conclusions, L275F and L299F in cytochrome b and SdhC-I88F are suspected to be responsible for the different patterns of sensitivity to QoI and for oxycarboxin-resistance in P. horiana, respectively.     

Molecular characterization of boscalid- and penthiopyrad-resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

BACKGROUND: Didymella bryoniae has a history of developing resistance to single‐site fungicides. A recent example is with the succinate‐dehydrogenase‐inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B^VHR) and highly resistant (B^HR) to boscalid respectively. Cross‐resistance studies with the new SDHI penthiopyrad showed that the B^VHR isolates were only highly resistant to penthiopyrad (B^VHR‐P^HR), while the B^HR isolates appeared sensitive to penthiopyrad (B^HR‐P^S). In this study, the molecular mechanism of resistance in these two phenotypes (B^VHR‐P^HR and B^HR‐P^S) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. RESULTS: A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B^S‐P^S), two B^VHR‐P^HR and one B^HR‐P^S isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild‐type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B^VHR‐P^HR and B^HR‐P^S variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B^VHR‐P^HR and B^HR‐P^S variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild‐type isolates. CONCLUSION: The genotype‐specific cross‐resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross‐resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management. Copyright © 2011 Society of Chemical Industry     

Detection of virulence to septoria tritici blotch (STB) resistance conferred by the winter wheat cultivar Cougar in the Irish Zymoseptoria tritici population and potential implications for STB control

Septoria tritici blotch (STB) caused by the fungal pathogen Zymoseptoria tritici continues to be the most economically destructive disease of winter wheat throughout Ireland. Due to the widespread development of fungicide resistance in the Irish Z. tritici population, integrated strategies to control STB are increasingly necessary. A key component of such strategies will be the deployment of winter wheat cultivars with improved levels of STB resistance. Unfortunately, due to the nature of Z. tritici, such resistances are at risk of being overcome by the pathogen. In late summer 2020, foci of STB were observed across a range of winter wheat cultivars under evaluation for recommendation in Ireland. Common amongst these was the cultivar Cougar in each of their pedigree. To determine if the foci observed in 2020 resulted from strains virulent to Cougar, isolate collections were established and virulence screens conducted on Cougar and a range of the cultivars currently under evaluation. These confirmed the presence of Cougar-virulent strains in the Irish Z. tritici population, and that this virulence affects not just Cougar, but also cultivars derived from it. Although the foci observed in 2020 were in both fungicide-untreated and -treated plots, there was no evidence that these strains are more sensitive or resistant to fungicides compared to the wider Irish Z. tritici population, with moderate resistance to the SDHIs and azoles dominating. Combined, the present study confirms the need to ensure a diversity of control measures for STB, including ensuring a range of STB resistances are used.     

Temporal and spatial field evaluations highlight the importance of the presymptomatic phase in supporting strong partial resistance in Triticum aestivum against Zymoseptoria tritici

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.     

Resistance of wheat pathogen Zymoseptoria tritici to DMI and QoI fungicides in the Nordic-Baltic region - a status

Septoria tritici blotch (STB) caused by the ascomycete Zymoseptoria tritici (Z. tritici) is currently the most prevalent foliar disease in wheat in the Nordic-Baltic region. Fungicide availability in this region differs greatly and is generally more limited than in other European regions. Monitoring of fungicide sensitivity is an essential tool to survey changes in fungal populations in order to react and be able to adapt recommendations for fungicide use. In this study the authors give an overview of the current situation of 14α-demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) sensitivity of Z. tritici from Scandinavia and the Baltic countries. A total of 985 isolates from the Nordic-Baltic region were investigated for EC₅₀ of DMI epoxiconazole and prothioconazole. Fungicide sensitivity remains at a high level with values ranging from 0.07 to 0.48 mg L^?1 for epoxiconazole and 1.17 to 9.47 mg L^?1 for prothioconazole. Point mutation I381V in the DMI target gene CYP51 was dominant throughout the region, but mutations D134G, V136A/C and S524T were also detected in the population in 2014. Screening for inserts in the CYP51 promoter region revealed that a ~ 1000 bp insert is predominant in the entire region. Only a single isolate was found in Denmark, harbouring the 120 bp insert, known to reduce fungicide sensitivity. Two Danish isolates which had elevated resistance levels were associated with an enhanced efflux. Significant differences were found across the area for the presence of G143A, conferring QoI resistance. As there is only limited access to results from this area, these findings can serve as reference for future fungicide sensitivity investigations and for evaluation of changes in the Northern European Z. tritici population.     

Evaluation of the susceptibility of modern,wild, ancestral,and mutational wheat lines to Septoria tritici blotch disease

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.     

Baseline sensitivity to proquinazid in Blumeria graminis f. sp. tritici and Erysiphe necator and cross‐resistance with other fungicides

BACKGROUND: Proquinazid is a new quinazolinone fungicide from DuPont registered in most European countries for powdery mildew control in cereals and vines. The aim of this paper is to present baseline sensitivity data in populations of Blumeria graminis f. sp. tritici EM Marchal and Erysiphe necator (Schw) Burr as well as results from cross‐resistance studies with other fungicides. RESULTS: Proquinazid exhibited a high intrinsic activity on B. graminis f. sp. tritici isolates at rates ranging from 0.000078 to 0.02 mg L^?1. Erysiphe necator isolates were comparatively less sensitive to proquinazid, with EC₅₀ values ranging from 0.001 to 0.3 mg L^?1. Proquinazid controlled equally well B. graminis f. sp. tritici isolates sensitive and resistant or less sensitive to tebuconazole, fenpropimorph, fenpropidin, cyprodinil and kresoxim‐methyl. A positive correlation (r = 0.617) between quinoxyfen and proquinazid sensitivities was found among 51 B. graminis f. sp. tritici isolates. Quinoxyfen‐resistant B. graminis f. sp. tritici isolates were slightly less sensitive to proquinazid than the quinoxyfen‐sensitive isolates; however, proquinazid remained much more active than quinoxyfen on these isolates. A stronger sensitivity relationship (r = 0.874) between proquinazid and quinoxyfen was found among 65 E. necator isolates tested in a leaf disc assay. The sensitivity values for proquinazid were significantly lower than those for quinoxyfen, confirming the higher intrinsic activity of proquinazid on both pathogens. CONCLUSION: Given the history of resistance development in powdery mildew and the observed sensitivity relationship with quinoxyfen, specifically in E. necator, we conclude that the risk of resistance developing to proquinazid might be influenced by the use of quinoxyfen. Based on these results, the authors recommend that proquinazid and quinoxyfen be managed together for resistance management. Copyright © 2009 Society of Chemical Industry     

Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK‐2A

BACKGROUND

Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK‐2A. Its mode of action and target site interactions have been investigated.

RESULTS

UK‐2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK‐2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK‐2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK‐2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross‐resistance to strobilurin, azole or benzimidazole fungicides.

CONCLUSION

Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK‐2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Isoflucypram,the first representative of a new succinate dehydrogenase inhibitor fungicide subclass: Its chemical discovery and unusual binding mode

Succinate dehydrogenase inhibitors (SDHIs) have played a crucial role in disease control to protect cereals as well as fruit and vegetables for more than a decade. Isoflucypram, the first representative of a newly installed subclass of SDHIs inside the Fungicide Resistance Action Committee (FRAC) family of complex II inhibitors, offers unparalleled long‐lasting efficacy against major foliar diseases in cereals. Herein we report the chemical optimization from early discovery towards isoflucypram and the first hypothesis of its altered binding mode in the ubiquinone binding site of succinate dehydrogenase. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Biological and molecular characterization of field isolates of <Emphasis Type="Italic">Alternaria alternata</Emphasis> with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC₅₀ values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.     

Fungicide resistance status and chemical control options for the brassica pathogen Pyrenopeziza brassicae

Pyrenopeziza brassicae causes leaf spot disease of Brassicaceae in Europe/Oceania (lineage 1) and North America (lineage 2). In Europe, fungicides currently used for disease management are sterol 14α-demethylase (CYP51) inhibitors (azoles), quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs); methyl benzimidazole carbamates (MBCs) are no longer applied. In this study, in vitro screening revealed European populations (collected 2018–2020) had shifted towards decreased azole sensitivity, but the North American population (2014–2016) was highly sensitive. Genotyping revealed CYP51 substitutions G460S or S508T were prevalent in European populations, often with a CYP51 promoter insert. Compared to wildtype CYP51 isolates, those with G460S plus an insert (44/46/151/210/302 bp) were c.25–32-fold and c.50-fold less sensitive to tebuconazole and prochloraz, respectively; those with S508T plus an insert (44/46/151/233 bp) were c.9–15-fold and c.25–40-fold less sensitive to tebuconazole and prochloraz, respectively. Selection for G460S (quantified via pyrosequencing) under different fungicide regimes was investigated in UK field trials, but G460S levels were high (c.76%) before treatment, so further selection during the trials was unclear. Despite the high G460S frequency and low disease pressure, yield data indicated measurable benefit for both azole- and non-azole-based programmes. In vitro screening against the MBC carbendazim showed European populations were predominantly moderately resistant/resistant; the North American population was sensitive. European and North American populations were sensitive to QoI (pyraclostrobin) and SDHI (penthiopyrad) fungicides. Results support an azole plus QoI/SDHI mixing partner for robust disease control and decreased risk of resistance, with continued sensitivity monitoring to ensure optimal strategies are deployed.     

Early molecular signatures of responses of wheat to Zymoseptoria tritici in compatible and incompatible interactions

Zymoseptoria tritici, the causal agent of septoria tritici blotch, a serious foliar disease of wheat, is a necrotrophic pathogen that undergoes a long latent period. Emergence of insensitivity to fungicides, and pesticide reduction policies, mean there is a pressing need to understand septoria and control it through greater varietal resistance. Stb6 and Stb15, the most common qualitative resistance genes in modern wheat cultivars, determine specific resistance to avirulent fungal genotypes following a gene‐for‐gene relationship. This study investigated compatible and incompatible interactions of wheat with Z. tritici using eight combinations of cultivars and isolates, with the aim of identifying molecular responses that could be used as markers for disease resistance during the early, symptomless phase of colonization. The accumulation of TaMPK3 was estimated using western blotting, and the expression of genes implicated in gene‐for‐gene interactions of plants with a wide range of other pathogens was measured by qRT‐PCR during the presymptomatic stages of infection. Production of TaMPK3 and expression of most of the genes responded to inoculation with Z. tritici but varied considerably between experimental replicates. However, there was no significant difference between compatible and incompatible interactions in any of the responses tested. These results demonstrate that the molecular biology of the gene‐for‐gene interaction between wheat and Zymoseptoria is unlike that in many other plant diseases, indicate that environmental conditions may strongly influence early responses of wheat to infection by Z. tritici, and emphasize the importance of including both compatible and incompatible interactions when investigating the biology of this complex pathosystem.     

Effect of azole fungicide mixtures,alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesized that combinations of azoles could slow the evolution of resistance. This work assessed the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre‐ and post‐treatment. Epoxiconazole and metconazole were applied as solo products, in alternation with each other, and as a pre‐formulated mixture. Full and half label doses were tested. Isolates were partially cross‐resistant to the two azoles, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0·1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.     

Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (>50% relative growth when using a spiral dilution gradient starting at 507 mg L^?1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.     

Measurement of infection efficiency of a major wheat pathogen using time-resolved imaging of disease progress

Infection efficiency is a key epidemiological parameter that determines the proportion of pathogen spores able to infect and cause lesions once they have landed on a susceptible plant tissue. In this study, an improved method to measure infection efficiency of Zymoseptoria tritici using a replicated greenhouse experiment is presented. Zymoseptoria tritici is a fungal pathogen that infects wheat leaves and causes septoria tritici blotch (STB), a major disease of wheat worldwide. A novel experimental setup was devised, where living wheat leaves were attached to metal plates, allowing for time-resolved imaging of disease progress in planta. Because lesions were continuously appearing, expanding and merging during the period of up to 3 weeks, daily measurements were necessary for accurate counting of lesions. Reference membranes were also used to characterize the density and spatial distribution of spores inoculated onto leaf surfaces. In this way, the relationship between the number of lesions and the number of viable spores deposited on the leaves was captured and an infection efficiency of about 4% was estimated from the slope of this relationship. This study provides a proof of principle for accurate and reliable measurement of infection efficiency of Z. tritici. The method opens opportunities for determining the genetic basis of the component of quantitative resistance that suppresses infection efficiency. This knowledge would improve breeding for quantitative resistance against STB, a control measure considered more durable than deployment of major resistance genes.