Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors

Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), a foliar wheat disease important worldwide. Succinate dehydrogenase inhibitors (SDHIs) have been used in cereals for effective control of STB for several years, but resistance towards SDHIs has been reported in several phytopathogenic fungi. Resistance mechanisms are target‐site mutations in the genes coding for subunits B, C and D of the succinate dehydrogenase (SDH) enzyme. Previous monitoring data in Europe indicated the presence of single isolates of Z. tritici with reduced SDHI sensitivity. These isolates carried mutations leading to amino acid exchanges: C‐T79N, C‐W80S in 2012; C‐N86S in 2013; B‐N225T and C‐T79N in 2014; and C‐V166M, B‐T268I, C‐N86S, C‐T79N and C‐H152R in 2015. The current study provides results from microtitre and greenhouse experiments to give an insight into the impact of different mutations in field isolates on various SDHIs. In microtitre tests, the highest EC₅₀ values for all tested SDHIs were obtained with mutants carrying C‐H152R. Curative greenhouse tests with various SDHIs confirmed the findings of microtitre tests that isolates with C‐H152R are, in general, controlled with lower efficacy than isolates carrying B‐T268I, C‐T79N and C‐N86S. SDHI‐resistant isolates of Z. tritici found in the field were shown to have cross‐resistance towards all SDHIs tested. So far, SDHI‐resistant isolates of Z. tritici have been found in low frequencies in Europe. Therefore, FRAC recommendations for resistance management in cereals, including a limited number of applications, alternation and combination with other MOAs, should be followed to prolong SDHI field efficacy.     

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.     

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.     

Pyraclostrobin reduces germ tube growth of QoI‐resistant Mycosphaerella graminicola pycnidiospores and the severity of septoria tritici blotch on winter wheat

The effect of the quinone outside inhibitors (QoI) azoxystrobin and pyraclostrobin on yields of winter wheat where QoI resistant Mycosphaerella graminicola isolates were dominant was investigated in field trials in 2006 and 2007. Pyraclostrobin significantly increased yields by 1·57 t ha^?1 in 2006 and 0·89 t ha^?1 in 2007 when compared to the untreated controls, while azoxystrobin only provided a significant increase of 1·28 t ha^?1 in 2006. These yield increases were associated with reduction in septoria tritici blotch (STB) development as determined by weekly disease assessments over a 7 week interval. The effect of pyraclostrobin on STB was studied in controlled environment experiments using wheat seedlings inoculated with individual M. graminicola isolates. Pyraclostrobin significantly reduced STB symptoms by up to 62%, whether applied 48 h pre‐ or post‐ inoculation with resistant M. graminicola isolates containing the cytochrome b mutation G143A. Extremely limited disease (<1%) was observed on similarly treated seedlings inoculated with an intermediately resistant isolate containing the cytochrome b mutation F129L, while no disease was observed on seedlings inoculated with a wild‐type isolate. Germination studies of pycnidiospores of M. graminicola on water agar amended with azoxystrobin or pyraclostrobin showed that neither fungicide inhibited germination of spores of resistant isolates containing the mutation G143A. However, pyraclostrobin significantly reduced germ tube length by up to 46% when compared with the untreated controls. Although the QoIs can no longer be relied upon to provide effective M. graminicola control, this study provides an insight into why QoIs still provide limited STB disease control and yield increases even in situations of high QoI resistance.     

Sources of resistance to septoria tritici blotch and implications for wheat breeding

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.     

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.     

Sensitivity to silthiofam, tebuconazole and difenoconazole of Gaeumannomyces graminis var. tritici isolates from China

BACKGROUND: Wheat take‐all caused by Gaeumannomyces graminis var. tritici (Ggt) has become an emerging threat to wheat production in the last few years. Silthiofam is very effective against Ggt, and recently it has been widely used for the control of take‐all in China. However, farmers have noted a decline in control efficacy with this compound in some wheat fields, suggesting that the pathogen may have developed resistance to silthiofam. RESULTS: Of the 66 Ggt isolates collected from different locations in China, 27 were resistant to silthiofam. There was no cross‐resistance between silthiofam and tecuconazole or difenoconazole. The effectiveness of silthiofam in controlling take‐all was compromised on wheat inoculated with silthiofam‐resistant isolates. Based on the DNA fingerprinting generated by microsatellite PCR, two predominant genetic clusters were found among these isolates and were clearly associated with the sensitivity to silthiofam. CONCLUSION: Silthiofam has a high risk in the development of resistance in Ggt. Tebuconazole and difenoconazole show great potential for control of take‐all on wheat. Results from this study provide useful information for take‐all control and the management of fungicide resistance. Copyright © 2012 Society of Chemical Industry     

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.     

Contributions of disease resistance and escape to the control of septoria tritici blotch of wheat

The contributions of disease escape and disease resistance to the responses of wheat to septoria tritici leaf blotch (STB) were analysed in a set of 226 lines, including modern cultivars, breeding lines and their progenitors dating back to the origin of scientific wheat breeding. Field trials were located in the important wheat-growing region of eastern England and were subject to natural infection by Mycosphaerella graminicola . STB scores were related to disease-escape traits, notably height, leaf spacing, leaf morphology and heading date, and to the presence of known Stb resistance genes and isolate-specific resistances. The Stb6 resistance gene was associated with a reduction of 19% in the level of STB in the complete set of 226 lines and with a 33% reduction in a subset of 139 lines of semidwarf stature. Greater plant height was strongly associated with reduced STB in the full set of lines, but only weakly in the semidwarf lines. Shorter leaf length was also associated with reduced STB, but, in contrast to earlier reports, lines with more prostrate leaves had more STB on average, probably because they tended to have longer leaves. Several lines, notably cvs Pastiche and Exsept, had low mean levels of STB which could not be explained by either escape traits or specific resistance genes, implying that they have unknown genes for partial resistance to STB.     

A review of the known unknowns in the early stages of septoria tritici blotch disease of wheat

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.     

Early stages of septoria tritici blotch epidemics of winter wheat: build‐up,overseasoning, and release of primary inoculum

Septoria tritici blotch (STB), caused by Mycosphaerella graminicola, is the most prevalent disease of wheat worldwide. Primary inoculum and the early stages of STB epidemics are still not fully understood and deserve attention for improving management strategies. The inoculum build‐up and overseasoning involves various fungal structures (ascospores, pycnidiospores, mycelium) and plant material (wheat seeds, stubble and debris; wheat volunteers; other grasses). Their respective importance is assessed in this review. Among the mechanisms involved in the early stages of epidemics and in the year‐to‐year disease transmission, infection by ascospores wind‐dispersed from either distant or local infected wheat debris is the most significant. Nevertheless, infection by pycnidiospores splash‐dispersed either from neighbouring wheat debris or from senescent basal leaves has also been inferred from indirect evidence. Mycosphaerella graminicola has rarely been isolated from seeds so that infected seed, although suspected as a source of primary inoculum for a long time, is considered as an epidemiologically anecdotal source. Mycosphaerella graminicola can infect a few grasses other than wheat but the function of these grasses as alternative hosts in natural conditions remains unclear. Additionally, wheat volunteers are suspected to be sources of STB inoculum for new crops. This body of evidence is summarized in a spatio‐temporal representation of a STB epidemic aimed at highlighting the nature, sources and release of inoculum in the early stages of the epidemic.     

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.     

Fungicide sensitivity of populations of wheat powdery mildew (Erysiphe graminis f.sp. Tritici) in Central Europe in 1993

In 1993 we observed the sensitivity of wheat powdery mildew populations (Erysiphe graminis DC f.sp. tritici Marchal) from the Czech Republic, Austria, Hungary and Slovakia to the fungicides triadimenol, tebuconazole, propiconazole, flutriafol and fenpropimorph. The highest resistance value was shown to triadimenol, which attained a mean resistance factor (MRF) of 29 (expressing how many times the population is more resistant than are standard sensitive isolates) in the mildew population from the Czech Republic. The mildew populations from eastern Slovakia and eastern Hungary, populations geographically isolated from the other populations, showed very high sensitivity to all fungicides tested. There was most sensitivity to fenpropimorph (smallest MRF values) compared with the other fungicides. Cross-resistance was established among all triazoles used, but not between triazoles and fenpropimorph. Sensitivity of wheat powdery mildew populations from Central Europe to these fungicides is considered adequate, and the development of resistance has shown a decreasing tendency in recent years.     

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     

Identification of isolate-specific and partial resistance to septoria tritici blotch in 238 European wheat cultivars and breeding lines

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.     

A gene in European wheat cultivars for resistance to an African isolate of Mycosphaerella graminicola

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 .     

小麦条锈病抗病遗传及菌源基地基因布局研究进展

小麦条锈病是世界范围内严重影响小麦生产安全的重要病害。我国是世界上最大的小麦条锈病流行区,自成独立的流行体系。培育和种植抗病品种是防治病害最有效的措施。然而,小麦品种对条锈病的抗性常常由于病菌新小种的产生而丧失,这既是一个重大科学问题,也是一个亟待研究解决的生产实际问题。如何有效、合理地利用小麦的抗病性,植病学家和育种学家进行了一个多世纪的研究与探索,提出了各种理论与策略,开展了各种实践与探索。该文就小麦抗条锈病遗传及其基因布局研究进展进行综述,主要包括抗性鉴定评价、抗病基因发掘与利用、数量抗性位点定位、抗病基因克隆与功能解析、近等基因系创建与应用,以及抗源创制、抗病生态育种和大区基因布局等,并对深入开展抗条锈病基因发掘与利用和大区基因布局进行展望,以期为抗病育种和病害持续治理提供参考。     

The wheat stem rust pathogen in the central region of the Russian Federation

A total of 387 isolates of Puccinia graminis f. sp. tritici (Pgt) collected in the central region of the Russian Federation from 2000 to 2009 was analysed with North American differential sets comprising 16 genotypes for samples of 2000–2006 and 20 genotypes for samples from 2007–2009. Forty‐five races were identified. The race composition of the local population underwent changes during this period. Race MKBT was the predominant race in the earlier years, but TKNT and TKNTF were in the majority later. During 2000–2009 there were no stem rust epidemics in the region. It was assumed that the local pathogen population cycled on wild grasses (including Elytrigia, Agropyron, Festuca, Dactylis, Phleum and Lolium spp.) and not only on wheat. The existence of host communities of wheat stem rust was supported by random amplified polymorphic DNA (RAPD) markers produced with high‐GC primers. The local population of Pgt was considered to be sexual based on the relatively high diversity of races isolated from various hosts and the absence of correlation between virulence attributes and molecular markers.     

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.