Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.     

Correlation of in planta endo‐beta‐1,4‐xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola

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.     

Evaluation of a predictive model for Mycosphaerella graminicola for economic and environmental benefits

A method was developed to evaluate crop disease predictive models for their economic and environmental benefits. Benefits were quantified as the value of a prediction measured by costs saved and fungicide dose saved. The value of prediction was defined as the net gain made by using predictions, measured as the difference between a scenario where predictions are available and used and a scenario without prediction. Comparable 'with' and 'without' scenarios were created with the use of risk levels. These risk levels were derived from a probability distribution fitted to observed disease severities. These distributions were used to calculate the probability that a certain disease induced economic loss was incurred. The method was exemplified by using it to evaluate a model developed for Mycosphaerella graminicola risk prediction. Based on the value of prediction, the tested model may have economic and environmental benefits to growers if used to guide treatment decisions on resistant cultivars. It is shown that the value of prediction measured by fungicide dose saved and costs saved is constant with the risk level. The model could also be used to evaluate similar crop disease predictive models.     

Genetic structure of Mycosphaerella graminicola populations in Iran

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.     

<Emphasis Type="Italic">Mycosphaerella graminicola</Emphasis> produces a range of cell wall-degrading enzyme activities <Emphasis Type="Italic">in vitro</Emphasis> that vary with the carbon source

In this study, two Mycosphaerella graminicola isolates produced a range of cell wall-degrading enzymes (CWDE) in vitro that can potentially degrade wheat cell walls. The influence of three carbon sources on CWDE in vitro production was tested: 1) 1 % galactose (w/v), 2) 1% wheat cell walls (w/v) and 3) a mixture of 1% galactose (w/v) and 1% wheat cell walls (w/v). Six major activities produced by both isolates were detected: xylanase, β-1,3-glucanase, polygalacturonase, cellulase, β-xylosidase and β-galactosidase. Time-course experiments showed that different levels of enzyme activities were obtained with isolates 323 and 94269. These activities levels varied also with the type of carbon source used. Principal Component Analysis showed that the enzyme activities are gathered into two groups. None of the activities of the first group was correlated to the activities of the second group. It also showed that the optimal medium that allowed the production of most of the major activities contained both galactose and wheat cell walls.     

Quantifying the effects of fungicides and disease resistance on greenhouse gas emissions associated with wheat production

A method is presented to quantify the net effect of disease management on greenhouse gas (GHG) emissions per hectare of crop and per tonne of crop produce (grain, animal feed, flour or bioethanol). Calculations were based on experimental and survey data representative of UK wheat production during the period 2004–06. Elite wheat cultivars, with contrasting yields and levels of disease resistance, were compared. Across cultivars, fungicides increased yields by an average of 1·78 t ha^?1 and GHG emissions were reduced from 386 to 327 kg CO₂ eq. t^?1 grain. The amount by which fungicides increased yield – and hence reduced emissions per tonne – was negatively correlated with cultivar resistance to septoria leaf blotch (Mycosphaerella graminicola, anamorph Septoria tritici). GHG emissions of treated cultivars were always less than those of untreated cultivars. Without fungicide use, an additional 0·93 Mt CO₂ eq. would be emitted to maintain annual UK grain production at 15 Mt, if the additional land required for wheat production displaced other UK arable crops/set aside. The GHG cost would be much greater if grassland or natural vegetation were displaced. These additional emissions would be reduced substantially if cultivars had more effective septoria leaf blotch resistance. The GHGs associated with UK fungicide use were calculated to be 0·06 Mt CO₂ eq. per annum. It was estimated that if it were possible to eliminate diseases completely by increasing disease resistance without any yield penalty and/or developing better fungicides, emissions could theoretically be reduced further to 313 kg CO₂ eq. t^?1 grain.     

Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control

This review summarises recent investigations into the molecular mechanisms responsible for the decline in sensitivity to azole (imidazole and triazole) fungicides in European populations of the Septoria leaf blotch pathogen, Mycosphaerella graminicola. The complex recent evolution of the azole target sterol 14α‐demethylase (MgCYP51) enzyme in response to selection by the sequential introduction of progressively more effective azoles is described, and the contribution of individual MgCYP51 amino acid alterations and their combinations to azole resistance phenotypes and intrinsic enzyme activity is discussed. In addition, the recent identification of mechanisms independent of changes in MgCYP51 structure correlated with novel azole cross‐resistant phenotypes suggests that the further evolution of M. graminicola under continued selection by azole fungicides could involve multiple mechanisms. The prospects for azole fungicides in controlling European M. graminicola populations in the future are discussed in the context of these new findings. Copyright © 2012 Society of Chemical Industry     

Resistance of wheat cultivars and breeding lines to septoria tritici blotch caused by isolates of Mycosphaerella graminicola in field trials

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.     

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.     

Presence of the Stb6 gene for resistance to septoria tritici blotch (Mycosphaerella graminicola) in cultivars used in wheat-breeding programmes worldwide

Fifty-two wheat cultivars and breeding lines, most of which have been used in breeding programmes worldwide, were tested for isolate-specific resistance to Mycosphaerella graminicola isolate IPO323, which interacts with the Stb6 gene 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 had Stb6 , a gene allelic to it or a gene closely linked to it. In 14 lines, resistance to IPO323 was controlled by Stb6 only. An exception was Kavkaz-K4500 L6.A.4., which has two genes for resistance to IPO323, one of which is Stb6 . The microsatellite marker Xgwm369 was used to examine genetic diversity in the region of the genome containing Stb6 , to which it is closely linked. Eleven alleles of Xgwm369 , 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 with Xgwm369 alleles, it was shown that Stb6 entered world wheat-breeding programmes on a minimum of six occasions, and possibly from as many as 11 sources. The presence of Stb6 in both European and Chinese landraces suggests that this gene has been present in cultivated wheat since the earliest times of agriculture.     

Relative importance of different types of inoculum to the establishment of Mycosphaerella graminicola in wheat crops in north‐west Europe

The contribution of wheat debris to the early stages of septoria leaf blotch epidemics was assessed in a 3‐year field experiment. First lesions were detected very early (December) in the case of an early sowing (mid‐October), showing that the first contamination could occur as soon as the seedlings emerge. The tested debris management options (chopped debris, removal of debris followed by tillage, or tillage in absence of debris) had a strong effect, although transient, on the epidemic dynamic: the more debris present on the soil surface, the more severe initial disease was. The magnitude of differences between treatments differed substantially between years. The relative production of pycnidiospores and ascospores was measured on the chopped debris. Peaks in pycnidiospore and ascospore production coincided in October–November. Both types of spores can be involved as primary inoculum in north‐west European conditions. The local amount of pycnidiospores available on debris in the field, estimated per square metre, was 1000‐fold the local ascospore production. Moreover, inoculum production was quantified on debris exposed to different environmental conditions. Autumnal conditions, characterized by moderate temperature with alternating wet and dry periods, were favourable for the production of both pycnidiospores and ascospores, as shown by the high inoculum production on debris exposed to field or outdoor conditions. By late autumn, the canopy became the most important source of pycnidiospores, and this period, characterized by the decreasing role of debris as a local source of inoculum compared to distant potential sources, can be considered as the end of the early epidemic stages.     

Identification and location of Stb9, a gene for resistance to septoria tritici blotch in wheat cultivars Courtot and Tonic

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.     

Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14 alpha-demethylation inhibitors in field isolates of Mycosphaerella graminicola

In France, as in many other European countries, Mycosphaerella graminicola (Fuckel) Schr?ter in Cohn (anamorph Septoria tritici), the causal agent of wheat leaf blotch, is controlled by foliar applications of fungicides. With the recent generalization of resistance to strobilurins (QoIs), reliable control is mainly dependent upon inhibitors of sterol 14 alpha-demethylation (DMIs). To date, strains with reduced sensitivity to DMIs are widespread, but disease control using members of this class of sterol biosynthesis inhibitors has not been compromised. In this study, sensitivity assays based on in vitro effects of fungicides towards germ-tube elongation allowed the characterization of seven DMI-resistant phenotypes. In four of them, cross-resistance was not observed between all tested DMIs; this characteristic concerned prochloraz, triflumizole, fluquinconazole and tebuconazole. Moreover, the highest resistant factors to most DMIs were found only in recent isolates; according to their response towards prochloraz, they were classified into two categories. Molecular studies showed that DMI resistance was associated with mutations in the CYP51 gene encoding the sterol 14 alpha-demethylase. Alterations at codons 459, 460 and 461 were related to low resistance levels, whereas, at position 381, a valine instead of an isoleucine, in combination with the previous changes, determined the highest resistance levels to all DMIs except prochloraz. Mutations in codons 316 and 317 were also found in some isolates exhibiting low resistance factors towards most DMIs.     

Resistance of wheat to septoria tritici blotch (Mycosphaerella graminicola) and associations with plant ideotype and the 1BL−1RS translocation

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.     

Genetic relationship between the adult plant resistance gene Lr12 and the complementary gene Lr31 for seedling resistance to leaf rust in common wheat

A resistant phenotype similar to that conferred in wheat by the complementary genes Lr27  +  Lr31 was produced in the progeny of intercrosses of cultivars carrying Lr27 and a line possessing Lr12 . This confirms that Lr12 is either completely linked with Lr31 or is the same gene. On the basis of these findings and that Lr31 is located on chromosome 4BS, it is concluded that Lr12 must also be located on 4BS. Adult-plant genetic tests confirm that the Australian wheat cultivar Timgalen carries Lr12 , and stocks with Lr12 alone were established from this cultivar.     

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.     

Differential seedling resistance to the eyespot pathogens,Oculimacula yallundae and Oculimacula acuformis,conferred by Pch2 in wheat and among accessions of Triticum monococcum

Eyespot is an economically important stem‐base disease of wheat caused by two fungal species: Oculimacula yallundae and Oculimacula acuformis. This study investigated the efficacy of two sources of resistance, viz. the genes Pch1, introgressed into hexaploid wheat from Aegilops ventricosa, and Pch2, identified in wheat cv. Cappelle Desprez, against O. yallundae and O. acuformis separately. In a series of seedling bioassays Pch1 was found to be highly effective against both species. Although Pch2 was found to confer resistance against both pathogen species, it was significantly less effective against penetration from O. yallundae than O. acuformis. Furthermore, a quantitative trait locus (QTL) analysis was not able to locate any resistance to O. yallundae on chromosome 7A of Cappelle Desprez. This has important implications for the use of Pch2 in commercial cultivars as it is necessary to have genes that confer resistance to both pathogens for effective eyespot control. In addition, a set of 22 T. monococcum accessions was screened for resistance to both O. yallundae and O. acuformis to identify potentially novel resistances and to assess the accessions for evidence of differential resistance to the eyespot species. Significant differences in resistance to the two pathogens were identified in four of these lines, providing evidence for differential resistance in T. monococcum. This study demonstrates that future screening for novel sources of eyespot resistance should investigate both pathogen species.