Virulence profile and genetic structure of a North Dakota population of Pyrenophora teres f. teres, the causal agent of net form net blotch of barley

A Pyrenophora teres f. teres population in North Dakota was analyzed for virulence variation and genetic diversity using 75 monospore isolates that were collected across a 4-year period (2004 to 2007) from two North Dakota State University agricultural experiment stations at Fargo and Langdon. Pathogenicity tests by inoculation onto 22 barley differential lines at seedling stage revealed 49 pathotypes, indicating a wide range of pathogenic diversity. Two-way analysis of variance of disease ratings revealed a significant difference in the virulence among isolates and in the resistance among barley lines, as well as in the interactions between the two. 'CI5791', 'Algerian', and 'Heartland' were three barley lines showing a high level of seedling resistance to all North Dakota isolates tested; however, many previously reported resistance genes have been overcome. Forty multilocus genotypes were identified from this set of isolates by genotyping at 13 simple-sequence repeat loci. High percentages of clonal cultures were detected in the samplings from 2005 and 2007 in Fargo and 2005 in Langdon. Using a clone-corrected sample set, the mean gene diversity (h) was estimated to be 0.58, approximately the same for both locations. The calculated Wright's F(ST) value is small (0.11) but was significantly >0, indicating a significant differentiation between the Fargo and Langdon populations. In the gametic disequilibrium test, only 3 of 78 possible pairwise comparisons over all isolates showed significant (P < 0.05) nonrandom association, suggesting a random mating mode. Our results suggest that the populations from the two locations are derived from a common source and undergo frequent recombination. This research provides important information for barley breeders regarding development and deployment of cultivars with resistance to net form net blotch in this region.     

Global Hierarchical Gene Diversity Analysis Suggests the Fertile Crescent Is Not the Center of Origin of the Barley Scald Pathogen Rhynchosporium secalis

A total of 1,366 Rhynchosporium secalis isolates causing scald on barley, rye, and wild barley (Hordeum spontaneum) were assayed for restriction fragment length polymorphism loci, DNA fingerprints, and mating type, to characterize global genetic structure. The isolates originated from 31 field populations on five continents. Hierarchical analysis revealed that more than 70% of the total genetic variation within regions was distributed within a barley field. At the global level, only 58% of the total genetic variation was distributed within fields, while 11% was distributed among fields within regions, and 31% was distributed among regions. A significant correlation was found between genetic and geographic distance. These findings suggest that gene flow is common at the local level while it is low between regions on the same continent, and rare between continents. Analyses of multilocus associations, genotype diversity, and mating type frequencies indicate that sexual recombination is occurring in most of the populations. We found the highest allele richness in Scandinavia followed by Switzerland. This suggests that R. secalis may not have originated at the center of origin of barley, the Fertile Crescent, nor in a secondary center of diversity of barley, Ethiopia.     

Adult plant resistance to powdery mildew (Erysiphe graminis)in three barley cultivars

The mechanism(s) of adult plant resistance (a.p.r.) to Erysiphe graminis f. sp. hordei were examined in two spring barley cultivars, Athos and Porthos, which possess similar identified mildew resistance genes and seedling resistance, but differential field resistance. These cultivars were compared with Golden Promise, a universally susceptible cultivar. Differential a.p.r. to a compatible race was detected as a reduction in the number and sporulation of colonies on later-formed Athos leaves. Golden Promise exhibited some a.p.r., but much less than either Athos or Porthos. Differential a.p.r. was expressed at several infection stages, viz. appressorial formation, penetration and sporulation. Epidermal hypersensitivity occurred infrequently in all leaf position/cultivar interactions. Papillae were associated with >90% of all penetration failures. The incidence of papillae in successful penetrations at 96 h was 20% on later-formed leaves of Athos compared to c. 60% on all other leaves.     

Identification of Molecular Genetic Markers in Pyrenophora teres f. teres Associated with Low Virulence on 'Harbin' Barley

ABSTRACT Two isolates of the barley net blotch pathogen (Pyrenophora teres f. teres), one possessing high virulence (0-1) and the other possessing low virulence (15A) on the barley cultivar Harbin, were crossed and the progeny of the mating were isolated. Conidia from cultures of the parent and progeny isolates were used as inoculum to determine the inheritance of virulence in the pathogen. Of the 82 progeny tested, 42 exhibited high virulence and 40 exhibited low virulence on 'Harbin' barley. The data support a model in which a single, major gene controls virulence in P. teres f. teres on this barley cultivar (1:1 ratio; chi(2) = 0.05, P = 0.83). Preparations of DNA were made from parental and progeny isolates, and the DNA was subjected to the random amplified polymorphic DNA (RAPD) technique in a search for molecular genetic markers associated with the virulence phenotype. Five RAPD markers were obtained that were associated in coupling with low virulence. The data indicate that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres.     

Breeding for Highly Fertile Isolates of Nectria haematococca MPVI that are Highly Virulent on Pea and In Planta Selection for Virulent Recombinants

ABSTRACT The heterothallic ascomycete Nectria haematococca mating population VI (anamorph Fusarium solani) is a broad host range pathogen. Field isolates of this fungus that are pathogenic on pea tend to be female sterile, of low fertility, and the same mating type (MAT-1), whereas female fertile isolates of either mating type that are highly fertile tend to be nonpathogenic on this plant. To facilitate genetic analysis of traits that may be important in the ability of N. haematococca to parasitize peas, a breeding project was undertaken to produce hermaphroditic isolates of each mating type that are highly fertile and highly virulent on peas. Although the association of high virulence on peas with female sterility and the MAT-1 mating type was not completely broken, isolates with high fertility and high virulence on peas were bred within two generations. Highly virulent progeny were also isolated by an alternative method in which pea plants were inoculated with a mixture of ascospores from a cross between two moderately virulent parents. Whereas all ascospores isolated without selection in planta had lower virulence than the parents, many isolates recovered from diseased tissue were more virulent than the parental isolates. Some of the recovered isolates were shown by restriction fragment length polymorphism analysis to be genetic recombinants of the parents, demonstrating that the pea tissue selected virulent recombinants. All highly virulent isolates tested had the ability to detoxify the pea phytoalexin pisatin, again showing a link between this trait and pathogenicity on the pea.     

Distribution of Mating Types and Diversity in Virulence of <Emphasis Type="Italic">Didymella rabiei</Emphasis> in Israel

The distribution of mating types and diversity in virulence of Didymella rabiei populations were studied in Israel from 1997 to 1999. Forty-one monoconidial D. rabiei isolates from 18 commercial fields distributed among all the chickpea production areas of the country were paired with MAT1-1 and MAT1-2 mating type tester isolates of D. rabiei. Both mating types were found in all chickpea production areas of the country. Of the 18 fields sampled, MAT1-1 was observed in 44%, and MAT1-2 in 88% of the sites. In some sites both mating types were present in close proximity, suggesting that sexual reproduction of the pathogen was feasible. The contribution of sexual reproduction of the fungus to virulence diversity was tested on detached leaves of six differential chickpea cultivars. Nine isolates were derived from different well separated foci (derived from ascospores as inoculum) and eight isolates were derived from a single, well defined infection focus (derived from sister conidia). In the analyses of variance the cultivar × isolate interaction showed no significant (P of F>0.09) effect on disease incidence; the chickpea cultivars differed significantly (P of F<0.0001) in their response to D. rabiei; and the isolate effect was highly significant (P of F = 0.0007) for the conidial population, but not significant (P of F>0.1) among isolates of the ascosporic population. Nevertheless, when comparing a cultivar at a time, the ascosporic and conidial populations did not differ significantly (P of F>0.1) in their virulence diversity. Virulence of 41 isolates collected from the different chickpea fields was tested on detached leaves of four Israeli cultivars that differ in their field response to D. rabiei. The cultivar × isolate interaction showed no significant effect (P of F = 0.95) on disease incidence. The main effects of cultivar and isolate on disease incidence were highly significant (P of F<0.0001). Accordingly, our data do not support the hypothesis that there is pathogenic specialization in the D. rabiei–C. arietinum pathosystem in Israel.     

玉蜀黍赤霉对小麦品种致病力的测定方法和致病力的分化

 玉蜀黍赤霉[Gibberella zeae(Schw) Petch]的子囊孢子、分生孢子和菌丝体对麦穗的致病力相同。以1-2个子囊孢子或分生孢子接种麦穗即能引起穗腐,将孢子的接种量增加到10个以上,侵染效率也能达100%。菌丝体接种的侵染速度要比孢子快。小麦扬花期离体接种小穗,记载病原菌侵入小穗轴和主穗轴的程度,是鉴定小麦品种抗扩展的可靠方法。
根据对在小麦感病品种矮秆早或抗病品种苏麦三号小穗接种测定的结果,全国各地具有代表性的56个菌株的致病力强弱不同。从强菌株F69分离到的37个单子囊孢子菌株其致病力有显著差异,但致病力的强弱不稳定。从同一个子囊分离到的单子囊孢子菌株,它们的致病力也有分化。菌株的致病力虽然有不稳定性,但不同品种的抗性还是明显的。试验证明玉蜀黍赤霉是同宗配合的,因此关于它的变异问题有待从各个方面进行深入研究。     

Racial diversity and complexity in regional populations of Erysiphe graminis f.sp. hordei in France over a 5-year period

The evolution of race patterns in three French regional populations of the barley powdery mildew pathogen Erysiphe graminis f.sp. hordei over a 5-year period showed rapid adaptation to newly introduced host resistance genes. In all three regions, the main change consisted of the replacement of initially abundant races by pathotypes differing markedly from them by their virulence gene combinations. This explained the increase in diversity during the first 3 years of the survey, when the second group of pathotypes became more common in the populations, and its subsequent decrease due to the decline of the first group of races. The mean number of virulence genes per isolate did not vary noticeably over time in the three populations, remaining at about four out of 12 genes tested. However, the distribution of the isolates into virulence complexity classes was greatly modified, fitting a binomial distribution by the end of the study, although significant deviations were apparent in the first 2 years (1986 and 1987). The data indicate that selection, migration and recombination are the most important factors shaping race structure and evolution in powdery mildew populations, and that mutation is of limited significance. No convincing evidence was obtained for the existence of stabilizing selection sensu Vanderplank as the mechanism limiting virulence complexity. Implications regarding spatial and temporal deployment of race-specific resistance genes to control powdery mildew are discussed.     

Detection of components of partial resistance to mildew (Erysiphe graminis f.sp. hordei) incorporated into advanced breeding lines of barley using measurement of fungal cell wall sterol

Characters for partial resistance to mildew ( Erysiphe graminis f.sp. hordei ) derived from primitive barley lines were tested and found to have been transmitted to F9 progeny of crosses with the susceptible cultivar Golden Promise with varying degrees of efficiency, and putative transgressive segregation was observed. Derived sterol extracts from mildew cell walls were used, in conjunction with infection frequency data, to quantify the mildew present, and this sterol was characterized as being of MW 470, probably (3.beta)-ergosta-5,24(28)dienol (C31.H54.0.SI SILANE).     

Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola

ABSTRACT Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat fields.     

Diversity and Complexity of Erysiphe graminis f.sp. hordei Collected from Barley Cultivar Mixtures or Barley Plots Treated with a Resistance Elicitor

Powdery mildew populations were analysed to determine the effects of a resistance elicitor and cultivar mixtures on genetic complexity and diversity. Isolations were made from a range of spring barley monocultures and mixtures in a field trial, and characterised for virulence and RAPD profile. In a second trial, isolates were taken from a single mixture from untreated and resistance elicitor-treated areas and from the components of the mixture in monoculture. The mildew population was not only highly heterogeneous for virulence characteristics, but also proved heterogeneous within pathotypes for molecular markers, indicating the major impact of sexual recombination on population structure and the lack of clonal dominance. Various diversity measurements were compared and the value of dissimilarity measurement for revealing genetic distance within a population was highlighted. There was a trend towards increasing complexity as the season progressed, but there was no consistent relationship between cultivar or mixture, disease control treatment, fertiliser treatment, replicate or position in trial, and pathogen genotype. Whilst the resistance elicitor did reduce mildew by 78% in the first trial, and there was no interaction with fertiliser level in its expression, control was substantially less in the second trial. There were no differences between mildew isolates from elicitor and control treatments. It was felt that more effective and consistent resistance elicitors need to be developed before it can be stated that they are unlikely to be eroded by selecting resistant or adapted mildew genotypes.     

Exploration and Exploitation Strategies of Powdery Mildew on Barley Cultivars with Different Levels of Nutrients

The mass fractal dimension (MFD) of colonies of mildew (Erysiphe graminis f.sp. hordei) growing on barley was calculated as a measure of their spatial structure. Despite the elongated shape of the colonies imposed by the leaf cellular structure, the MFD remained constant with scale. The mildew MFD differed on different cultivars of barley, and was greater on leaves produced under higher nutrient level indicating a physiological component. Lower MFD values correspond with the thin spreading growth associated with exploration strategies and higher values correspond to the denser, more branched structure associated with exploitation of the substrate. Cultivars showing exploration strategies induced by resistance expression responded to increased nutrient levels more than those expressing little resistance such as Golden Promise.     

Host responses to fungal penetration in Evysiphe graminis f.sp. hordei infections in barley

Resistance mechanisms restricting penetration and establishment were investigated in an incompatible interaction using an avirulent race CC/3 (BMV 1 + 4) of Erysiphe graminis f.sp hordei on barley cv. Athos (BMR 2 + 5) and a compatible interaction using the universal susceptible cv. Golden Promise (BMR 0). In both interactions: (i) auto-fluorescence of the host cell wall occurred adjacent to the primary germ tube tip after 4 h, and by 10 h near to the appressorial germ tube; (ii) strongly fluorescing papillae developed after 12 h, being frequently associated with failed penetrations; and (iii) only 30% of the attempted penetrations from appressorial lobes resulted in an incipient haustorium at 16 h. Hypersensitive reactions occurred in cells with an incipient haustorium in 30% of appressorial penetrations on Athos, but only in 4% of those on Golden Promise. Twenty per cent of hypersensitively reacting cells in Athos appeared dead by 14 h using trypan blue (membrane exclusion test), compared with 5% for neutral red, suggesting that plasma membrane damage is an early event in the hypersensitive response. Haustorial death was associated with host cell death, but did not precede it; appressorial death occurred 2-4 h after that of the host cell.     

四川西南部小麦白粉菌群体毒性及遗传多样性分析

为明确四川西南部白粉菌群体毒性及其遗传变异情况,本文将2011年采自四川西南部的小麦白粉病标样进行单孢子堆分离纯化,共获得48个白粉菌菌株,并将其按采集地划分为4个地理群体。利用28份已知抗白粉病基因材料测定了群体的毒性频率,并运用SRAP分子标记技术探讨了其遗传多样性。毒性测定结果表明,白粉菌群体毒性较强,群体间毒性结构存在差异。供试群体对Pm1、Pm3a、Pm3b、Pm3d、Pm5a、Pm6、Pm19的平均毒性频率达50%以上,而Pm13、Pm XBD、Pm5b、Pm2+6、Pm5+6抗性保持良好,平均毒性频率在10%以下。群体间毒性遗传距离与地理距离之间有一定的相关性,但未达到显著水平。用筛选出的10对SRAP引物共获得160个清晰、稳定的扩增位点,多态性频率为50.63%;白粉菌群体遗传多样度(h)、Shannon信息指数(I)分别为0.198 8、0.322 7,遗传变异主要源于群体内部。群体间基因流数值均在6.50以上,说明四川西南部白粉菌群体间菌源迁移频繁,基因交流较为充分。M antel Test分析表明SRAP标记解释的群体遗传多样性与地理距离、毒性多样性间的相关性未达到显著水平。     

Development of apothecia of the eyespot pathogen Tapesia on cereal crop stubble residue in England

A reservoir of infection of Tapesia yallundae may exist after harvest in bases of cereal stems due to the presence of apothecia capable of discharging infective ascospores. Apothecia of T. yallundae developed in a seasonal pattern on winter barley inoculated with the pathogen, with maximum numbers of apothecia produced on stubble 5–7 months after harvest. A similar pattern of development was observed on infected winter wheat. However, the peak in numbers of mature apothecia was observed 2 months later than in winter barley. Apothecia capable of discharging ascospores were present for up to 6 months on stubble. Apothecia of T. acuformis were not detected on spring or winter barley, or spring or winter wheat stubble, despite inoculation of growing crops with isolates of compatible mating type.     

Pathogenicity of Rhynchosporium secalis isolates from Norway on 30 cultivars of barley

The pathogenic variability of the barley scald fungus, Rhynchosporium secalis , in central Norway was examined in 1994. The climate in this region is usually cold and wet during the growing season of spring barley. Leaf blotch is prevalent and causes significant yield losses. Forty-two isolates of the fungus, from naturally infected spring barley in four counties, were differentiated into 32 pathotypes by the standard differential set for R. secalis . All pathotypes were complex and had virulence for nine to 22 differentials. The cultivar Osiris was resistant to all isolates tested. The cultivars C.I.8162, Hudson, Atlas 46 and C.I.3515 were resistant to the majority of the isolates. Several differentials with various resistance genes were susceptible to up to 100% of the isolates. Isolates were derived from local cultivars with no known resistance genes, suggesting that R. secalis populations in central Norway are characterized by a high degree of seemingly unnecessary pathogenicity. Because of the great variability and complexity of the pathotypes, traditional breeding methods using single major genes are not likely to be effective in central Norway.     

Effect of early infection on pathotype frequencies in barley powdery mildew (Blumeria graminis f.sp. hordei) populations in field plots

A field experiment with barley powdery mildew ( Blumeria graminis f.sp. hordei ) was designed in order to study how the time of arrival of inoculum in the field influenced pathotype frequencies in the resulting populations. Three isolates belonging to pathotypes that were absent or rare in the local aerial inoculum were used to inoculate field plots of winter barley cv. Plaisant. Two successive inoculations with different combinations of the three isolates were performed with an approximately two-generation delay, and frequencies of inoculated pathotypes were assessed four and nine generations after the first inoculation. Pathotypes of the first inoculated isolates generally persisted throughout the period of sampling; this is described as an 'early arrival' effect. During the epidemics the inoculated isolates were not replaced by isolates from the natural airborne inoculum. Pathotype frequencies depended mainly on the time of arrival of inoculum in the plot, but frequencies also depended on the isolate that had been inoculated. The most frequent isolate, GL1, belonged to the clonal lineage dominant in powdery mildew populations on winter barley in the north of France. These results confirmed that the composition of a powdery mildew population in a field is largely determined by the composition of the initial inoculum.     

Barley Powdery Mildew Populations on Volunteers and Changes in Pathotype Frequencies During Summer on Artificially Inoculated Field Plots

Pathotype frequencies in barley powdery mildew populations were assessed in artificially inoculated barley plots. The field experiment was organised in eighteen 3 m × 3 m plots with different inoculum compositions, obtained by sequential inoculation with three isolates, gl-1, gl-2 and gl-3, shortly after seedling emergence. In the conidia populations before summer, pathotypes corresponding to the inoculated isolates were detected at frequencies in the range 11–42% for GL1, 0–14% for GL2 and 2–34% for GL3. On the volunteers appearing after harvest these three pathotypes were observed at lower frequencies: 0–37% for GL1, 0–12% for GL2 and 0–23% for GL3. The overall ranking of GL1, GL2 and GL3 frequencies was thus the same before and after summer. The populations on volunteers were influenced by both sexual and asexual populations present in the same field at the end of the previous growing season. However, at the small-scale level no simple correlation was found between the frequencies in the conidia populations on volunteers and those in the airborne population, or the conidia populations on the crop before summer, or the calculated expected frequencies in populations of ascospores. During the summer survival, chance events may also have a large influence on pathotype frequencies leading to a high variation between repeated events of transition from the crop to the volunteers.     

Persistence of Complex Virulences in Populations of Phytophthora infestans in Western Washington

ABSTRACT Isolates of Phytophthora infestans, collected from bittersweet, hairy nightshade, petunia, potato, potato vine, and tomato in western Washington, 1998 to 1999, were evaluated for virulence complexity as well as mating type, metalaxyl insensitivity, allozymes of glucose-6-phosphate isomerase and peptidase, and DNA fingerprint with the RG57 probe. Results were compared with those from similar collections made in the same region during the 1990s. Generally, virulence complexity was high for most of the isolates regardless of year, genotype, or host. No marked shift in virulence complexity was evident for the populations studied, and unnecessary virulences were maintained. During 1998 and 1999, isolates of the US-8 and US-11 genotypes had 4 or more virulence factors. US-8 isolates averaged 8.2 and 9.3, whereas US-11 isolates averaged 5.4 and 6.3 virulence factors. The frequency of US-8 isolates that were sensitive to metalaxyl ranged from 5% in 1998 to 72% in 1999. All of the US-11 isolates tested in 1998 and 1999 were insensitive to metalaxyl. From 1996 to 1999 on potato, the recovery of US-8 increased, whereas the recovery of US-11 decreased. No evidence of new genotypes or sexual recombination was found. Western Washington was a desirable location for screening germ plasm for durable resistance to late blight due to the high frequency and persistence of complex virulences.     

Both Mating types of Phaeosphaeria (anamorph Stagonospora) nodorum are Present in Western Australia

Phaeosphaeria (anamorph Stagonospora) nodorum is the most serious fungal pathogen of wheat in the West Australian (WA) wheat belt and is a diallelic heterothallic loculoascomycete. Its population genetics has received considerable attention. A recent study, which sampled isolates from diverse locations worldwide, has indicated that the mating-type idiomorph MAT1-1 is considerably more frequent than MAT1-2 in many populations. To investigate this, we developed PCR primers that amplify each idiomorph. In a sample of 23 isolates cultured from ascospores collected in the field, nine amplified DNA with the MAT1-1 primers and 14 amplified DNA with the MAT1-2 primers. The virulence of a MAT1-2 isolate was comparable with MAT1-1 isolates. Although these sample sizes are small, we suggest that this result is consistent with the presence of equal numbers of both mating types in populations of ascospores in WA.