Cell wall-degrading enzymes produced in vitro by isolates of Phaeosphaeria nodorum differing in aggressiveness

The relationships between in vitro production of cell wall-degrading enzymes and aggressiveness of three Phaeosphaeria nodorum isolates were investigated. When grown in liquid medium containing 1% cell wall from wheat leaves as the carbon source, the isolates secreted xylanase, α-arabinosidase, β-xylosidase, polygalacturonase, β-galactosidase, cellulase, β-1,3-glucanase, β-glucosidase, acetyl esterase and butyrate esterase. Time-course experiments showed different levels of enzyme production and different kinetics between isolates. A highly aggressive isolate produced more xylanase, cellulase, polygalacturonase and butyrate esterase than did the two weakly aggressive isolates. Xylanase was the most active polymer-degrading enzyme produced, suggesting a key role during pathogenesis by P. nodorum .     

Mating type distribution and genetic structure are consistent with sexual recombination in the Swedish population of Phaeosphaeria nodorum

Mating type ratios and SSR marker analysis were used to study the genetic structure of Phaeosphaeria nodorum , the causal agent of glume blotch in wheat. The study was based on leaf collections in five fields located in different regions in Sweden. In total 302 isolates of P. nodorum were obtained from 203 sampling sites (including eight ascospore isolates). Three strong indications of sexual recombination were found: (i) the two mating types were present at a 1:1 ratio; (ii) the genetic structure was diverse, with many unique genotypes, and 69 of the 93 genotypes were only found once; and (iii) random association of alleles indicated that genetic recombination was frequent. However, asexual reproduction could not be excluded since identical genotypes were found within the fields. The fungal population had experienced a demographic bottleneck, as indicated by a low ratio of number of alleles to microsatellite size range ( M -value) of 0·5.     

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.     

SnToxA,SnTox1, and SnTox3 originated in Parastagonospora nodorum in the Fertile Crescent

The centre of origin of the globally distributed wheat pathogen Parastagonospora nodorum has remained uncertain because only a small number of isolates from the Fertile Crescent were included in earlier population genetic and phylogeographic studies. We isolated and genetically analysed 193 P. nodorum strains from three naturally infected wheat fields distributed across Iran using 11 neutral microsatellite loci. Compared to previous studies that included populations from North America, Europe, Africa, Australia, and China, the populations from Iran had the highest genetic diversity globally and also exhibited greater population structure over smaller spatial scales, patterns typically associated with the centre of origin of a species. Genes encoding the necrotrophic effectors SnToxA, SnTox1, and SnTox3 were found at a high frequency in the Iranian population. By sequencing 96 randomly chosen Iranian strains, we detected new alleles for all three effector genes. Analysis of allele diversity showed that all three effector genes had higher diversity in Iran than in any population included in previous studies, with Iran acting as a hub for the effector diversity that was found in other global populations. Taken together, these findings support the hypothesis that P. nodorum originated either within or nearby the Fertile Crescent with a genome that already encoded all three necrotrophic effectors during its emergence as a pathogen on wheat. Our findings also suggest that P. nodorum was the original source of the ToxA genes discovered in the wheat pathogens Phaeosphaeria avenaria f. sp. tritici 1, Pyrenophora tritici-repentis, and Bipolaris sorokiniana.     

Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead's Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2·5, 2·1 and 0·6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2·5 or 0·5 mm sulphate. Both pathogens infected less frequently at 2·5 mm sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4·5 at 2·5 mm sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance.     

Mating Relationships between Isolates of Phaeosphaeria nodorum, (anamorph Stagonospora nodorum) from Geographical Locations

Mating types of Phaeosphaeria nodorum isolates (from North Africa, North America, Australia, Europe and Near East) were determined in laboratory conditions. Although both mating types were found, MAT1-1 and MAT1-2 were not evenly distributed among the isolates. Sexual compatibility pairings with standard mating type testers revealed that 56 of 101 isolates could be assigned to MAT1-1 and 5 to MAT1-2. Although the teleomorph occurred in different countries, the two mating types were observed only among isolates collected from France, Great Britain, Germany and Morocco. For Morocco, where no P. nodorum pseudothecia have been reported, this is the first report of the existence of the two opposite mating types. The remaining 40 isolates could not be designated to a specific mating type. However, in 17 crosses, pseudothecial initials or sterile pseudothecia were observed on wheat straw after two months. The implications of the predominance of one mating type are discussed.     

Frequency of Phaeosphaeria nodorum, the Sexual Stage of Stagonospora nodorum, on Winter Wheat in North Carolina

ABSTRACT Ascocarps of Phaeosphaeria nodorum, which causes Stagonospora nodorum blotch (SNB) of wheat, have not been found by others in the eastern United States despite extensive searches. We sampled tissues from living wheat plants or wheat debris in Kinston, NC, each month except June from May to October 2003. Additional wheat samples were gathered in Kinston, Salisbury, and Plymouth, NC, in 2004 and 2005. For the 3 years, in all, 2,781 fruiting bodies were dissected from the wheat tissues and examined microscopically. Fruiting bodies were tallied as P. nodorum pycnidia or ascocarps, "unknown" (not containing spores, potentially P. nodorum or other fungi), or "other fungi." In the 2003 sample, asco-carps of P. nodorum were present each month after May at a frequency of 0.8 to 5.4%, and comprised a significantly higher percentage of fruiting bodies from wheat spikes than of those from lower stems and leaves. Ascocarps also were found at frequencies <10% in some wheat debris samples from 2004 and 2005. Analysis of the nucleotide sequences of internally transcribed spacer regions of 18 genetically distinct North Carolina isolates from 2003 suggested that all were P. nodorum, not the morphologically similar P. avenaria f. sp. triticea. Neither the 18 isolates from 2003 nor a set of 77 isolates derived from 2004 Kinston leaf samples gave reason to suspect a mating-type imbalance in the larger P. nodorum population (P >/= 0.4). We conclude that, in North Carolina, sexual reproduction plays a role in initiation of SNB epidemics and the creation of adaptively useful genetic variability, although its relative importance in structuring this population is uncertain.     

Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in Ontario isolates of Sclerotinia homoeocarpa

Four hundred and thirty-five isolates of Sclerotinia homoeocarpa from eight populations in southern Ontario were tested for sensitivity to the demethylation-inhibiting (DMI) fungicides, propiconazole, myclobutanil, fenarimol and tebuconazole. The isolates were collected in summer 1994 just prior to legal DMI fungicide use on turfgrass in Ontario. There were wide variations in sensitivities, and seven of the eight populations were very sensitive to the fungicides. Based on mean EC₅₀ and the distribution of DMI sensitivity, one population near the U.S. border was suspected of having been previously exposed to DMI fungicide. Pairwise comparisons of EC₅₀ values for the different fungicides showed low to moderate correlations between fungicides. EC₅₀ values of myclobutanil and propiconazole had the best correlation, followed by the pair of tebuconazole and fenarimol. Other pairwise comparisons were not statistically significant except for a barely significant relationship between EC₅₀ values of myclobutanil and tebuconazole. For field populations of plant pathogens, cross-resistance to different DMI fungicides may not be as strong as conventionally thought. The data collected here will allow comparison to subsequent years to look for detectable shifts in S. homoeocarpa sensitivity to DMI fungicides as they become more frequently used in Ontario.     

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α‐demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI‐resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real‐time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC₅₀ results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post‐inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch. Copyright © 2011 Society of Chemical Industry     

Genetic structure of Phaeosphaeria nodorum populations in the north-central and midwestern United States

Stagonospora nodorum blotch, caused by Phaeosphaeria nodorum, is considered one of the most destructive foliar diseases of wheat in the United States. However, relatively little is known about the population biology of this fungus in the major wheat-growing regions of the central United States. To rectify this situation, 308 single-spore isolates of P. nodorum were analyzed from 12 populations, five from hard red spring wheat cultivars in Minnesota and North Dakota and seven from soft red winter wheat in Indiana and Ohio. The genetic structure of the sampled populations was determined by analyzing polymorphisms at five microsatellite or simple-sequence repeat (SSR) loci and the mating type locus. Although a few clones were identified, most P. nodorum populations had high levels of gene (H(S) = 0.175 to 0.519) and genotype (D = 0.600 to 0.972) diversity. Gene diversity was higher among isolates collected from spring wheat cultivars in North Dakota and Minnesota (mean H(S) = 0.503) than in those from winter wheat cultivars in Indiana and Ohio (H(S) = 0.269). Analyses of clone-corrected data sets showed equal frequencies of both mating types in both regional and local populations, indicating that sexual recombination may occur regularly. However, significant gametic disequilibrium occurred in three of the four populations from North Dakota, and there was genetic differentiation both within and among locations. Genetic differentiation between the hard red spring and soft red winter wheat production regions was moderate (F(ST) = 0.168), but whether this is due to differences in wheat production or to geographical variation cannot be determined. These results suggest that sexual reproduction occurs in P. nodorum populations in the major wheat-growing regions of the central United States, and that geographically separated populations can be genetically differentiated, reflecting either restrictions on gene flow or selection.     

Sensitivity of Drechslera teres and Septoria nodorum to sterol-biosynthesis inhibitors

The sensitivity of 282 isolates ofSeptoria nodorum was tested on prochloraz, propiconazole and guazatine, and of 129 isolates ofDrechslera teres on prochloraz, propiconazole, imazalil and triadimefon. There was a great variation in sensitivity between isolates particularly at relatively low concentrations of the fungicides. However, none of the isolates was considered resistant towards the sterol biosynthesis inhibitors tested. Some isolates ofS. nodorum grew on concentrations of guazatine that may present difficulties in controlling them under practical conditions.Samenvatting Isolaten vanSeptoria nodorum (n=282) werden getoetst op gevoeligheid voor prochloraz, propiconazool en guazatine en vanDrechslera teres (n=129) voor prochloraz, propiconazool, imazalil en triadimefon. Er waren grote verschillen in de gevoeligheid van de isolaten, in het bijzonder bij lage concentraties van de fungiciden. Geen van de isolaten kan echter als resistent tegen sterolbiosynthese-remmers worden opgevat. Het feit, dat sommige van de isolaten vanS. nodorum zich ontwikkelden op media die hoge concentraties guazatine bevatten, kan echter duiden op moeilijkheden bij de bestrijding onder praktische omstandigheden.     

High Genetic Similarity Among Populations of Phaeosphaeria nodorum Across Wheat Cultivars and Regions in Switzerland

ABSTRACT Phaeosphaeria nodorum was sampled from nine wheat fields across a 30-km transect representing three geographical regions in Switzerland to determine the scale of genetic differentiation among subpopulations. Three different wheat cultivars were sampled three times to determine whether differences in host genotype correlated with differences among corresponding pathogen populations. Seven restriction fragment length polymorphism (RFLP) loci and one DNA fingerprint were assayed for each of the 432 isolates in the collection. DNA fingerprints differentiated 426 unique genotypes. Though absolute differences were small, five RFLP loci exhibited significant differences in allele frequencies across the nine sub-populations. Gene diversity within all subpopulations was high (H(T) = 0.51), but only 3% of the total genetic variation was distributed among the nine subpopulations. When subpopulations were grouped according to geographical region or host cultivar, less than 1% of the genetic variation was distributed among groups, suggesting widespread gene flow and the absence of pathogen adaptation to specific wheat cultivars. Tests for gametic equilibrium within subpopulations and across the entire Swiss population supported the hypothesis of random mating.     

丙环唑对水稻恶苗病菌(Fusarium fujikuroi)生物活性研究

 丙环唑是一种具有广谱活性的三唑类杀菌剂。本研究首次采用菌丝生长速率法建立了浙江省绍兴市、江苏省淮安市和黑龙江省尚志市不同地区的80株水稻恶苗病原菌(Fusarium fujikuroi)对丙环唑的敏感性基线,测定了丙环唑对水稻恶苗病菌的生理影响和对水稻的安全性。结果表明: 丙环唑对水稻恶苗病菌菌丝生长的有效抑制中浓度(EC₅₀值)范围在0.029 8~0.211 0 μg·mL^-1,EC₅₀平均值为(0.106 7 ± 0.004 4) μg·mL^-1。水稻恶苗病菌经0.1 μg·mL^-1(EC₅₀平均值)或3 μg·mL^-1(EC₉₀平均值)丙环唑处理后,分生孢子产量显著降低,细胞膜通透性显著升高;3 μg·mL^-1丙环唑不影响水稻恶苗病菌细胞核的分布与定位,但可以使细胞壁缢缩,菌丝顶端膨大,破坏水稻恶苗病菌细胞膜和细胞器。丙环唑浓度为不超过200 μg·mL^-1时,对水稻种子发芽率和株高无显著影响,同时可显著增加水稻鲜重;当浓度为500 μg·mL^-1时,对水稻种子发芽率、株高和鲜重均无显著性影响;当浓度达到1 000 μg·mL^-1时,发芽率显著降低但株高和鲜重无显著性差异;水稻种子发芽长度随丙环唑浓度升高而降低。本研究明确了丙环唑对水稻恶苗病菌的生物活性和对水稻的安全性,为丙环唑防治水稻恶苗病提供了指导,并进一步加深对丙环唑作用机理的认识。     

Structural Characterisation of the Interaction between Triticum aestivum and the Dothideomycete Pathogen Stagonospora nodorum

The interaction between Stagonospora nodorum and a susceptible wheat cultivar was investigated using a range of microscopic techniques. Germination of pycnidiospores occurred approximately 3 h after making contact with the leaf surface and was followed by attempted penetration 8–12 h later. Penetration was observed through stomata and also directly through periclinal and anticlinal epidermal cell walls. Penetration down the anticlinal cell walls appeared to occur without a differentiated penetrating structure whilst structures identified as either lateral appressoria or hyphopodia were typically present when penetrating over a periclinal cell wall. Once inside the leaf, the fungus continued to grow for the next 4–5 days colonising all parts of the leaf except the vascular bundles. Only in the later phase of the infection was total host cell collapse apparent. Evidence of polyphenolic compounds was observed. The infection cycle was completed within 7 days as indicated by sporulation on the leaf surface. These results have allowed us to understand how the fungus physically interacts with the leaf and will help the overall understanding of the infection process.     

黄瓜霜霉病菌对嘧菌酯的敏感基线研究

从山东、河北、武汉等未使用过甲氧基丙烯酸酯类杀菌剂(strobilurins)的地区采集19个黄瓜霜霉病菌 Pseudoperonospora cubensis 样本,采用叶盘漂浮法室内测定新药剂嘧菌酯(azoxystrobin)对该病菌的毒力,其EC50值范围为11.72×10-5~9.340×10-3 μg/mL。在19个菌系中选择最为敏感的河北定兴菌系进行重复测定,将其EC50平均值2.317×10-4 μg/mL 确定为黄瓜霜霉病菌对嘧菌酯的敏感基线。     

Variation in cultural characteristics, pathogenicity, vegetative compatibility and electrophoretic karyotype within field populations of Stagonospora nodorum

Isolates of Stagonospora nodorum , the cause of septoria leaf and glume blotch of wheat, were sampled intensively from single fields in two consecutive years and compared for growth rate, colony morphology, pathogenicity, vegetative compatibility and electrophoretic karyotype. The two populations were highly variable for all characters, even though the isolates in each population originated from a small geographical area. Studies of vegetative compatibility within each population indicated the presence of many genotypes and suggested that clonal spread was very limited. Individual lesions contained a single genotype and therefore, presumably, originated from a single unit of inoculum. However, adjacent lesions, even on the same leaf, frequently contained different genotypes. Ten isolates from population II possessed six different karyotypes, demonstrating that polymorphism for this fundamental feature of the genome occurs within field populations. The population structure revealed by these studies supports the hypothesis that ascospores play a major role in the epidemiology of the disease. On this basis, the observed variation in all the characters, including karyotype, can be explained by recombination during sexual reproduction.