Molecular Mapping of the Stb4 Gene for Resistance to Septoria tritici Blotch in Wheat

ABSTRACT Breeding wheat for resistance is the most effective means to control Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici). At least eight genes that confer resistance to STB in wheat have been identified. Among them, the Stb4 locus from the wheat cv. Tadinia showed resistance to M. graminicola at both seedling and adult-plant stages. However, no attempt has been made to map the Stb4 locus in the wheat genome. A mapping population of 77 F10 recombinant-inbred lines (RILs) derived from a three-way cross between the resistant cv. Tadinia and the susceptible parent (Yecora Rojo x UC554) was evaluated for disease resistance and molecular mapping. The RILs were tested with Argentina isolate I 89 of M. graminicola for one greenhouse season in Brazil during 1999, with an isolate from Brazil (IPBr1) for one field season in Piracicaba (Brazil) during 2000, and with Indiana tester isolate IN95-Lafayette-1196-WW-1-4 in the greenhouse during 2000 and 2001. The ratio of resistant:susceptible RILs was 1:1 in all three tests, confirming the single-gene model for control of resistance to STB in Tadinia. However, the patterns of resistance and susceptibility were different between the Indiana isolate and those from South America. For example, the ratio of RILs resistant to both the Indiana and Argentina isolates, resistant to one but susceptible to the other, and susceptible to both isolates was approximately 1:1:1:1, indicating that Tadinia may contain at least two genes for resistance to STB. A similar pattern was observed between the Indiana and Brazil isolates. The gene identified with the Indiana tester isolate was assumed to be the same as Stb4, whereas that revealed by the South American isolates may be new. Bulked-segregant analysis was used to identify amplified fragment length polymorphism (AFLP) and microsatellite markers linked to the presumed Stb4 gene. The AFLP marker EcoRI-ACTG/MseI-CAAA5 and microsatellite Xgwm111 were closely linked to the Stb4 locus in coupling at distances of 2.1 and 0.7 centimorgans (cM), respectively. A flanking marker, AFLP EAGG/ M-CAT10, was 4 cM from Stb4. The Stb4 gene was in a potential supercluster of resistance genes near the centromere on the short arm of wheat chromosome 7D that also contained Stb5 plus five previously identified genes for resistance to Russian wheat aphid. The microsatellite marker Xgwm111 identified in this study may be useful for facilitating the transfer of Stb4 into improved cultivars of wheat.     

Partial Resistance to Septoria Tritici Blotch (Mycosphaerella graminicola) in Wheat Cultivars Arina and Riband

ABSTRACT Partial resistance to Septoria tritici blotch (STB) and its inheritance were investigated in a doubled-haploid population of a cross between cvs. Arina and Riband. The former has good partial resistance whereas the latter is susceptible. In adult plant trials in polytunnels, STB disease scores were negatively correlated with heading date. Resistance was not specific to any of the three fungal isolates used in these tests. A quantitative trait locus (QTL) for partial resistance to STB was identified in Riband on chromosome 6B and is named QStb.psr-6B-1. No QTL controlling a major part of the Arina resistance was identified, suggesting that its resistance may be dispersed and polygenic. There was no correlation between the lines' mean disease scores at the seedling and adult stages, implying that partial resistance to STB is developmentally regulated. Seedling resistance to the isolate IPO323 was isolate-specific and controlled by a single gene in Arina, probably allelic with the Stb6 gene in cv. Flame that confers resistance to the same isolate. The implications of these results for wheat breeding programs are discussed.     

Wheat Leaf Rust Uredospore Production on Adult Plants: Influence of Leaf Nitrogen Content and Septoria tritici Blotch

ABSTRACT Leaf rust uredospore production and lesion size were measured on flag leaves of adult wheat plants in a glasshouse for different lesion densities. We estimated the spore weight produced per square centimeter of infected leaf, per lesion, and per unit of sporulating area. Three levels of fertilization were applied to the plants to obtain different nitrogen content for the inoculated leaves. In a fourth treatment, we evaluated the effect of Septoria tritici blotch on leaf rust uredospore production. The nitrogen and carbon content of the spores was unaffected or marginally affected by lesion density, host leaf nitrogen content, or the presence of Mycosphaerella graminicola on the same leaf. In leaves with a low-nitrogen content, spore production per lesion was reduced, but lesion size was unaffected. A threshold effect of leaf nitrogen content in spore production was however, evident, since production was similar in the medium- and high-fertilizer treatments. In leaves inoculated with M. graminicola and Puccinia triticina, the rust lesions were smaller and produced fewer spores. The relationships among rust lesion density, lesion size, and uredospore production were fitted to a model. We determined that the density effect on spore production resulted mainly from a reduction in lesion size, the spore production per unit of sporulating surface being largely independent of lesion density. These results are consistent with those obtained previously on wheat seedlings. The main difference was that the sporulation period lasted longer in adult leaves.     

Elucidation of Septoria tritici x Wheat Interactions Using GUS-Expressing Isolates

ABSTRACT Isolate ISR398 of Septoria tritici (which produces none to few pycnidia on the wheat cv. Seri 82 and high coverage on cv. Shafir) and isolate ISR8036 (which is virulent on both cultivars) were genetically cotrans-formed using the selectable marker gene hph, which confers resistance to hygromycin B (hygB), and the reporter gene uidA, encoding beta-glucuronidase (GUS). Most of the genetically transformed isolates (98.8%) produced similar pycnidial coverage on seedlings of 'Seri 82' and 'Shafir' as the two wild-type isolates. Southern analysis of 25 randomly selected hygB(R)GUS(+) transformants probed with the uidA sequence revealed multiple insertion sites. GUS activity was determined fluorimetrically by measuring the conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone (MU). The high GUS-expressing transformants 398D97 and 8036E27 were used to elucidate fungal development within inoculated leaf tissue by using GUS activity to estimate the fungal proteins content in planta. Increase in fungal biomass was recorded in 'Shafir' inoculated with the GUS-expressing transformants 398D97 and 8036E27 following a 12-day latent period. A 15-day latent period was recorded in 'Seri 82' inoculated with 8036E27, whereas an 18-day latent period was recorded on 'Seri 82' inoculated with 398D97 and the two mixtures 398D97 + ISR8036 and ISR398 + 8036E27. The rate of fungal development and the estimated level of fungal proteins at the pycnidia maturation stage was high in leaves of 'Shafir' and moderate to low on 'Seri 82', even in cases in which no significant differences were recorded in pycnidial coverage. An endogenous capacity to hydrolyze beta-1,4-D-glucuronidase was recorded in leaves inoculated with wild-type isolates. The latent periods in MU production of the uidA-expressing transformants mimicked those recorded for the wild-type isolates. However, at all stages, the levels of MU produced in wheat inoculated with wild-type isolates were markedly lower than those produced by GUS-expressing transformants. The mode of interaction (compatible or incompatible) determined the onset of the induction, rate, and level of enzyme production.     

Studies on the Interaction Between Septoria tritici and Stagonospora nodorum in Wheat

Interactions between Stagonospora nodorum and Septoria tritici were studied. Results from a detached glume experiment indicated that the interaction may be isolate-dependent, as it was shown that the interaction between the two pathogens may be beneficial or antagonistic depending on the isolate of each pathogen present. The number of spores produced by both pathogens was significantly greater when an aggressive isolate of S. tritici was mixed with a non-aggressive isolate of S. nodorum, whereas the number of spores produced by both pathogens was significantly less when two non-aggressive isolates were mixed. There was a significant reduction in disease level when S. tritici was applied prior to S. nodorum, compared to vice versa in the growth chamber. Results from growth chamber and field studies showed that S. nodorum produced significantly more spores when both pathogens were present together. It is concluded that S. tritici has a stimulatory effect on spore production by S. nodorum. However, there was a reduction of S. tritici spores observed in the dual inoculation treatments, suggesting that S. nodorum inhibits S. tritici.     

A Gene-for-Gene Relationship Between Wheat and Mycosphaerella graminicola, the Septoria Tritici Blotch Pathogen

ABSTRACT Specific resistances to isolates of the ascomycete fungus Mycosphaerella graminicola, which causes Septoria tritici blotch of wheat, have been detected in many cultivars. Cvs. Flame and Hereward, which have specific resistance to the isolate IPO323, were crossed with the susceptible cv. Longbow. The results of tests on F1 and F2 progeny indicated that a single semidominant gene controls resistance to IPO323 in each of the resistant cultivars. This was confirmed in F3 families of Flame x Longbow, which were either homozygous resistant, homozygous susceptible, or segregating in tests with IPO323 but were uniformly susceptible to another isolate, IPO94269. None of 100 F2 progeny of Flame x Hereward were susceptible to IPO323, indicating that the resistance genes in these two cultivars are the same, closely linked, or allelic. The resistance gene in cv. Flame was mapped to the short arm of chromosome 3A using microsatellite markers and was named Stb6. Fifty-nine progeny of a cross between IPO323 and IPO94269 were used in complementary genetic analysis of the pathogen to test a gene-for-gene relationship between Stb6 and the avirulence gene in IPO323. Avirulence to cvs. Flame, Hereward, Shafir, Bezostaya 1, and Vivant and the breeding line NSL92-5719 cosegregated, and the ratio of virulent to avirulent was close to 1:1, suggesting that these wheat lines may all recognize the same avirulence gene and may all have Stb6. Together, these data provide the first demonstration that isolate-specific resistance of wheat to Septoria tritici blotch follows a gene-for-gene relationship.     

Yield Reduction in Wheat in Relation to Leaf Disease From Yellow (tan) Spot and Septoria Nodorum Blotch

Yellow or tan spot (caused by Pyrenophora tritici-repentis) and septoria nodorum blotch (caused by Phaeosphaeria nodorum) occur together and are a constraint to wheat yields in Australia. Recently, higher crop yields and lower fungicide costs have made fungicides an attractive management tool against these diseases. Yield-loss under different rates of progress of yellow spot and septoria nodorum blotch was examined in four experiments over three years to define the relationship between disease severity and yield. In these experiments, differences in disease were first promoted by inoculations either with P. tritici-repentis-infected stubble or aqueous spore suspensions of P. nodorum. Disease progress was further manipulated with foliar application of fungicide. The pattern of disease development varied in each year under the influence of different rainfall patterns. The inoculation and fungicide treatments produced differences in disease levels after flag leaf emergence. The infection of yellow spot or septoria nodorum blotch caused similar losses in grain yield, ranging from 18% to 31%. The infection by either disease on the flag or penultimate leaf provided a good indication of yield-loss. Disease severity on flag leaves during the milk stage of the crop or an integration of disease as area under the disease progress curve on the flag leaves based on thermal time explained more than 80% variance in yield in a simple regression model. The data provided information towards the development of disease management strategies for the control of septoria nodorum blotch and yellow spot.     

Genotypic Diversity of the Wheat Leaf Blotch Pathogen Mycosphaerella Graminicola (anamorph) Septoria Tritici in Germany

The population structure and genotypic diversity of Mycosphaerella graminicola from six natural field populations in Germany were studied with molecular markers. To reveal the potential effects of plant host resistance on the pathogen population, hierarchical samples were taken from susceptible and resistant cultivars. A total of 203 single spore isolates was subjected to molecular marker analysis using the amplified fragment length polymorphism technique (AFLP). Among the 203 isolates analyzed, 142 different multilocus haplotypes (MLH) were identified revealing a high degree of genotypic diversity of the M. graminicola population. On average, a F _ST value of 0.04 was found, indicating a low genetic differentiation with only 4% of the genetic variation between the local populations but leaving 96% of the genetic variation within the populations. According to the low F _ST value, a high migration rate of Nm 12 was found. The observed high within-population diversity, and the significant migration between populations, prevented genetic isolation and differentiation of putative geographically separated populations. Furthermore, plant host resistance had no obvious effect on the population structure and diversity of M. graminicola. Genotypic variability can be attributed to sexual recombination which appears to have a considerably larger influence on the population structure. Gene flow on this scale could have significant implications for plant breeding and fungicide spraying programmes.     

Effects of Wheat Cultivar Mixtures on Epidemic Progression of Septoria Tritici Blotch and Pathogenicity of Mycosphaerella graminicola

ABSTRACT The effects of host genotype mixtures on disease progression and pathogen evolution are not well understood in pathosystems that vary quantitatively for resistance and pathogenicity. We used four mixtures of moderately resistant and susceptible winter wheat cultivars naturally inoculated with Mycosphaerella graminicola to investigate impacts on disease progression in the field, and effects on pathogenicity as assayed by testing isolate populations sampled from the field on greenhousegrown seedlings. Over 3 years, there was a correspondence between the mixtures' disease response and the pathogenicity of isolates sampled from them. In 1998, with a severe epidemic, mixtures were 9.4% less diseased than were their component pure stands (P = 0.0045), and pathogen populations from mixtures caused 27% less disease (P = 0.085) in greenhouse assays than did populations from component pure stands. In 1999, the epidemic was mild, mixtures did not reduce disease severity (P = 0.39), and pathogen populations from mixtures and pure stands did not differ in pathogenicity (P = 0.42). In 2000, epidemic intensity was intermediate, mixture plots were 15.2% more diseased than the mean of component pure stands (P = 0.053), and populations from two of four mixtures were 152 and 156% more pathogenic than the mean of populations from component pure stands (P = 0.043 and 0.059, respectively). Mixture yields were on average 2.4 and 6.2% higher than mean component pure-stand yields in 1999 and 2000, respectively, but the differences were not statistically significant. The ability of mixtures challenged with M. graminicola to suppress disease appears to be inconsistent. In this system, host genotype mixtures evidently do not consistently confer either fitness benefits or liabilities on pathogen populations.     

In vitro production of pycnidia by Septoria tritici

The in vitro production of pycnidia bySeptoria tritici was examined on six media reported to induce the formation of fruiting bodies. Among 26 freshly isolated cultures from various parts of the world, consistent differences in growth type were found which were only partially influenced by nutritional and environmental conditions. Cultures with yeast-like growth produced hardly any pycnidia or pseudopycnidia, while cultures with intermediate or mycelial growth types produced them frequently. Incubation in continuous darkness induced intermediate to mycelial growth types rather than yeast-like growth types in some cultures, and concomitantly the production of more pycnidia. Potato-dextrose agar induced intermediate to mycelial growth types and production of (pseudo)pycnidia more often than V8 agar and wheat leaf extract agar, which had previously been reported to be especially beneficial to (pseudo) pycnidium formation byS. tritici. Isolates with a consistently yeast-like growth type, producing (virtually) no fructifications under any of the experimental conditions, were slightly stimulated to form pseudopycnidia on water agar supplemented with sterile pieces of maize, wheat or carnation leaves.     

Modeling of Relationships Between Weather and Septoria tritici Epidemics on Winter Wheat: A Critical Approach

ABSTRACT Two models for predicting Septoria tritici on winter wheat (cv. Riband) were developed using a program based on an iterative search of correlations between disease severity and weather. Data from four consecutive cropping seasons (1993/94 until 1996/97) at nine sites throughout England were used. A qualitative model predicted the presence or absence of Septoria tritici (at a 5% severity threshold within the top three leaf layers) using winter temperature (January/February) and wind speed to about the first node detectable growth stage. For sites above the disease threshold, a quantitative model predicted severity of Septoria tritici using rainfall during stem elongation. A test statistic was derived to test the validity of the iterative search used to obtain both models. This statistic was used in combination with bootstrap analyses in which the search program was rerun using weather data from previous years, therefore uncorrelated with the disease data, to investigate how likely correlations such as the ones found in our models would have been in the absence of genuine relationships.     

The Occurrence of Mycosphaerella graminicola and its Anamorph Septoria tritici in Winter Wheat during the Growing Season

The disease septoria tritici blotch of wheat is initiated by ascospores of the teleomorph Mycosphaerella graminicola or pycnidiospores of the anamorph Septoria tritici. We report for the first time the presence of the teleomorph, M. graminicola, in Denmark. With the objective of elucidating the importance of the teleomorph for the development of septoria tritici blotch, data on the occurrence of fruit bodies of the anamorph (pycnidia) and the teleomorph (pseudothecia) stages were collected over three growing seasons. Pseudothecia were present in the springs, however, high numbers of pseudothecia compared to pycnidia were not observed until July, too late to influence the epidemic. On an individual leaf layer, pycnidia were observed well before pseudothecia. As the leaves aged, progressively higher proportions of fruit bodies were observed to be pseudothecia. The period from the appearance of pycnidia to detection of pseudothecia was estimated as 29–53 days. At harvest, high proportions of sporulating fruit bodies in the crop were pseudothecia, suggesting that the primary source of inoculum for new emerging wheat crops in autumn is likely to be ascospores.     

Control of Stagonospora nodorum and Septoria tritici in Wheat by Pre-treatment with Drechslera teres, a Non-host Pathogen

A field study is described which explored the possibility of controlling Stagonospora nodorum and Septoria tritici on wheat using a barley pathogen, Drechslera teres. Pre-treatment of wheat cv. Hussar flag leaves with D. teres resulted in a significant reduction in disease caused by S. nodorum and S. tritici, resulting in a significant increase in grain yield. When cv. Brigadier leaves were treated with D. teres prior to inoculation with S. nodorum there was an initial increase in disease expression whilst D. teres had no effect on symptoms produced by S. tritici on cv. Brigadier. There was significantly less disease on leaves of cvs. Hussar and Brigadier pre-treated with D. teres prior to inoculation with an equal mixture of S. nodorum and S. tritici compared to plants pre-treated with water. It is concluded that D. teres and other non-host pathogens show potential as biological control agents for S. nodorum and S. tritici.     

不同耕作措施下春小麦应对干旱胁迫的生理响应

为了解春小麦在不同耕作措施下应对干旱胁迫的能力强弱,对不同耕作措施下春小麦叶片的光合生理特性、叶绿素含量、脯氨酸含量以及丙二醛(MDA)含量进行了测定分析。结果表明:经过8年的轮作后,与传统耕作(T)相比,免耕+秸秆覆盖(NTS)、免耕无覆盖(NT)和传统耕作+秸秆还田(TS)3种保护性耕作方式均能不同程度地提高拔节期、孕穗期、灌浆期春小麦叶片净光合速率、蒸腾速率、气孔导度以及叶绿素含量。与保护性耕作的3个处理相比,在春小麦生长的各生育期脯氨酸含量和丙二醛(MDA)含量均表现为保护性耕作的3个处理低于传统耕作,其中NTS处理最低。说明保护性耕作措施能有效缓解作物受到的干旱胁迫;而传统耕作措施下情况反之。     

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the -tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with -tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.