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.     

Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonase

Eight Mycosphaerella graminicola isolates were investigated for correlations between pathogenicity and the in vitro production of cell wall-degrading enzymes. Isolate pathogenicity was evaluated in terms of lesion and production of pycnidia in wheat leaves. Additionally, the isolates were compared over time for their ability to produce in vitro significant levels of xylanase (EC 3·2·1·8), β-xylosidase (EC 3·2·1·37), β-1,3-glucanase (EC 3·2·1·6), cellulose (EC 3·2·1·4) and polygalacturonase (EC 3·2·1·15) activities when grown in a liquid medium. Correlation tests and principal component analysis revealed a significant correlation between the in vitro production of xylanase and pectinase and pathogenicity components. Xylanase was correlated to necrosis frequency ( r  = 0·795), β-xylosidase was correlated to the mean of the lesion length ( r  = −0·787), whereas polygalacturonase was correlated to the time when 50% of the leaves contained a lesion ( r  = 0·776), the lesion frequency ( r  = 0·646) and the time when 50% of the leaves showed pycnidia ( r  = −0·711). The results suggest that these two groups of cell wall-degrading enzymes are therefore likely to be key determinants of pathogenicity in M. graminicola .     

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.     

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.     

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.     

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.     

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.     

Microscopic analysis of the effect of azoxystrobin treatments on Mycosphaerella graminicola infection using green fluorescent protein (GFP)‐expressing transformants

Green fluorescent protein (GFP)‐expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha^?1) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post‐penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola. The contribution of post‐penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed. © 2001 Society of Chemical Industry     

Cultivar‐dependent partial resistance and associated defence mechanisms in wheat against Zymoseptoria tritici

Septoria tritici blotch caused by the fungus Zymoseptoria tritici is one of the most devastating foliar diseases of wheat. Knowledge regarding mechanisms involved in resistance against this disease is required to breed durable resistances. This study compared the expression of defence and pathogenicity determinants in three cultivars in semicontrolled culture conditions. The most susceptible cultivar, Alixan, presented higher necrosis and pycnidia density levels than Altigo, the most resistant one. In Premio, a moderately resistant cultivar, necrosis developed as in Alixan, while pycnidia developed as in Altigo. In noninfectious conditions, genes coding for PR1 (pr1), glucanase (gluc) and allene oxide synthase (aos) were constitutively expressed at a higher level in both Altigo and Premio than in Alixan, while chitinase2 (chit2), phenylalanine ammonia‐lyase (pal), peroxidase (pox2) and oxalate oxidase (oxo) were expressed at a higher level in Premio only. Except for aos, all genes were induced in Alixan during the first steps of the symptomless infection phase. Only pox2, oxo, gluc and pal genes in Altigo and pal, chs and lox genes in Premio were up‐regulated at some time points. Basal cultivar‐dependent resistance against Z. tritici could therefore be explained by various gene expression patterns rather than high expression levels of given genes. During the necrotrophic phase, Z. tritici cell wall‐degrading enzyme activity levels were lower in Altigo and Premio than in Alixan, and were associated more with pycnidia than with necrosis. Similar tissue colonization occurred in the three cultivars, suggesting an inhibition of the switch to the necrotrophic lifestyle in Altigo.     

Transfer of the β‐tubulin gene of Botrytis cinerea with resistance to carbendazim into Fusarium graminearum

BACKGROUND: Resistance to carbendazim and other benzimidazole fungicides in Botrytis cinerea (Pers. ex Fr.) and most other fungi is usually conferred by mutation(s) in a single chromosomal β‐tubulin gene, often with several allelic mutations. In Fusarium graminearum Schwade, however, carbendazim resistance is not associated with a mutation in the corresponding β‐tubulin gene. RESULTS: The β‐tubulin gene conferring carbendazim resistance in B. cinerea was cloned and connected with two homologous arms of the β‐tubulin gene of F. graminearum by using a double‐joint polymerase chain reaction (PCR). This fragment was transferred into F. graminearum via homologous double crossover at the site where the β‐tubulin gene of F. graminearum is normally located (the β‐tubulin gene of F. graminearum had been deleted). The transformants were confirmed and tested for their sensitivity to carbendazim. CONCLUSION: The β‐tubulin gene conferring carbendazim resistance in B. cinerea could not express this resistance in F. graminearum, as transformants were still very sensitive to carbendazim. Copyright © 2010 Society of Chemical Industry     

Studies on the Infection Process of Fusarium Culmorum in Wheat Spikes: Degradation of Host Cell Wall Components and Localization of Trichothecene Toxins in Infected Tissue

After single spikelet inoculation, the infection process of Fusarium culmorum and spread of fungal hyphae in the spike tissues were studied by scanning and transmission electron microscopy. While hyphal growth on outer surfaces of the spike was scanty and no successful penetration was observed, the fungus developed a dense mycelium on the inner surfaces and effectively invaded the lemma, glume, palea and ovary by penetration pegs. During the inter- and intracellular spreading of the fungus, marked alterations in the host tissues were observed, including degeneration of cytoplasm, cell organelles, and depositions of electron dense material between cell wall and plasmalemma. Ultrastructural studies revealed that host cell walls in proximity of the penetration peg and in contact with hyphae were less dense or transparent which suggested that cell wall degrading enzymes were involved in colonisation of host tissues by fungal hyphae. Enzyme- and immunogold-labelling investigations confirmed involvement of extracellular enzymes, that is cellulases, xylanases and pectinases, in degradation of cell wall components. Localization studies of trichothecenes indicated that toxins could be detected in host tissues at an early stage of infection.     

Evidence that wheat cultivars differ in their ability to build up inoculum of the take‐all fungus,Gaeumannomyces graminis var. tritici,under a first wheat crop

The effect of wheat cultivar on the build‐up of take‐all inoculum during a first wheat crop was measured after harvest using a soil core bioassay in field experiments over five growing seasons (2003–2008). Cultivar differences in individual years were explored by analysis of variance and a cross‐season Residual Maximum Likelihood (REML) variance components analysis was used to compare differences in those cultivars present in all years. Differences between cultivars in the build‐up of inoculum were close to or at significance in two of the five trial years (2004 P < 0·05; 2006 P < 0·07), and current commercially listed cultivars were represented at both extremes of the range. In 2007 and 2008, when environmental conditions were most favourable for inoculum build‐up, differences were not significant (P < 0·3). In 2005 the presence of Phialophora spp. at the trial site restricted the build‐up of take‐all inoculum under all cultivars. The cross season REML variance components analysis detected significant differences (range: 3·4–47·8% roots infected in the soil core bioassay; P < 0·01) between the nine cultivars present in all years (excluding 2005). This is the first evidence of relatively consistent differences between hexaploid wheat cultivars in their interactions with the take‐all fungus, and this could give an indication of those cultivars that could be grown as a first wheat crop, in order to reduce the risk of damaging take‐all in a second wheat crop. This phenomenon has been named the take‐all inoculum build‐up (TAB) trait.