Two Indigenous Berberis Species From Spain Were Confirmed as Alternate Hosts of the Yellow Rust Fungus Puccinia striiformis f. sp. tritici.

Puccinia striiformis f. sp. tritici, which causes yellow (or stripe) rust on wheat, is a macrocyclic and heteroecious fungus. In this study, we investigated whether Berberis vulgaris subsp. seroi and B. vulgaris subsp. australis, which are indigenous in Spain, may serve as alternate hosts for P. striiformis f. sp. tritici. Wheat leaves bearing telia of an isolate of P. striiformis f. sp. tritici were harvested and used to inoculate plants of both barberry subspecies. Pycnia were observed on the adaxial side of the leaves from 10 days after inoculation (dai). Following successful fertilization, aecia were observed on the abaxial side of the leaves from 16 dai. At 27 dai, barberry leaves bearing aecia were detached and used to inoculate susceptible wheat seedlings of cultivar Morocco. Uredinia were observed on wheat seedlings from 12 days after aeciospore exposure. Eighty-three single lesions were recovered from individual wheat leaves, of which 43 were genotyped using 19 P. striiformis f. sp. tritici simple sequence repeat markers (SSR). In total, 19 multilocus genotypes (MLGs) were identified among the 43 progeny isolates. The SSR genotyping confirmed that all 43 isolates were derived from the parental isolate. Seven heterozygous SSR markers showed segregation among the progenies, whereas none of the 12 homozygous markers resulted in segregation. These results demonstrated that B. vulgaris subspp. seroi and australis can serve as alternate hosts for P. striiformis f. sp. tritici, which may result in novel virulence combinations that can have a detrimental impact on wheat production. Although P. striiformis f. sp. tritici has not been detected on these barberry species in nature, this study highlights the importance of rust surveillance in barberry areas where suitable conditions for completion of the sexual life cycle may be present.

Worldwide population structure of the wheat rust fungus Puccinia striiformis in the past.

Puccinia striiformis is a basidiomycete causing yellow rust on wheat. The availability of historic samples of this pathogen from the 'Stubbs collection' enabled us to investigate past population structure and temporal dynamics on a global scale. A set of 212 single genotype urediniospore isolates, representing samples collected from five continents between 1958 and 1991, were genotyped using 19 polymorphic microsatellite markers. The population genetic analyses revealed the existence of seven genetic groups in the past worldwide P. striiformis population. This genetic grouping generally corresponded with geographical sample origin except for the Middle East, where six of the seven genetic groups were represented. The presence of many genetic groups in the Middle Eastern population reflected a low differentiation from the populations in East Africa (FST=0.052) and in South Asia (FST=0.064). A high diversity and recombinant population structure was observed in China and South Asia, while a clonal population structure was observed in NW Europe, East Africa and the Mediterranean region. The high genetic diversity in the Himalayan region supported recent studies suggesting a putative center of diversity for P. striiformis in this area. Four of the 89 multilocus genotypes detected were resampled in different geographical regions suggesting long-distance migration in the past. Comparison of the past populations with more recent ones, represented by 309 isolates mainly collected between 2001 and 2009, revealed temporal divergence for all populations except for Northwest Europe. Overall, we observed a clear subdivision within the worldwide population structure of P. striiformis and migration in the past.

Rust Fungi Forming Aecia on Berberis spp. in Sweden.

Barberry (Berberis spp.) hosts the aecial stage of several rust species, including Puccinia graminis, which causes stem rust on grasses and cereals. The aecial stage of this pathogen has received less attention because it is not as economically important compared with the uredinial and telial stages. The main objective of this study was to identify and describe the rust species that were found on Berberis spp. collected in different parts of Sweden. A morphological study, including spore measurements and aecia descriptions, was conducted, as well as DNA sequence analyses (using the internal transcribed spacer region and the EF1-α gene). Based on spore and aecia morphology as well as the genetic analyses, three different taxa could be distinguished on barberry: P. graminis f. sp. avenae, P. graminis f. sp. tritci/secalis, and P. arrhenatheri. The genetic analysis revealed little or no differentiation between P. graminis f. sp. tritici and P. graminis f. sp. secalis and, thus, this group of samples was denominated P. graminis f. sp. tritici/secalis. Aecial morphology may be used to differentiate between different taxa. In particular, examination of aecial cross-sections may be used to distinguish between P. graminis f. sp. avenae and P. graminis f. sp. tritici/secalis. A clear differentiation in the mode of growth on barberry was also found; P. arrhenatheri always appeared systemic whereas P. graminis always appeared localized.

TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals.

Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also showed some correlation to 3ADON especially in wheat and triticale. Selected wheat and barley samples from 1957 to 2000 showed low amounts of F. graminearum and F. culmorum in general but with a dominance of the 3ADON genotype. 15ADON was not detected in these samples, except for very low amounts in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON genotype in 2003 and following years correspond well with the occurrence of F. graminearum and indicates that the 15ADON genotype was introduced along with F. graminearum around 2000. The amounts of the 3ADON and 15ADON genotypes correlated well with the total amount of DON whereas the amounts of NIV genotype correlated well with the amount of NIV in wheat and triticale but not in barley where the results indicate that Fusarium poae may also contribute to the NIV content.

Fusarium head blight of cereals in Denmark: species complex and related mycotoxins.

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.

Universally Primed Polymerase Chain Reaction Alleles and Internal Transcribed Spacer Restriction Fragment Length Polymorphisms Distinguish Two Subgroups in Botrytis aclada Distinct from B. byssoidea.

ABSTRACT Fifty-one isolates representing the four Botrytis spp. associated with onion neck rot were clustered by unweighted pair group method with arithmetic mean based on universal-primed polymerase chain reaction (UP-PCR) fingerprints. Bootstrap analysis of the consensus phenogram clearly demonstrated five strong clusters among the four Botrytis spp.: B. cinerea (C), B. squamosa (S), B. byssoidea (B), and B. aclada (AI and AII). Subdivision of the 30 B. aclada isolates, AI (14) and AII (16), from Europe, Egypt, North America, and Japan was further supported by restriction analysis of the internal transcribed spacer of the ribosomal genes and spore size measurements. Gene diversities (H) among AI and AII isolates were very low (0.007 and 0.043, respectively). A likelihood ratio chi-square test (G(2)) of Nei's coefficient of genetic differentiation (G(ST)) showed that both B. aclada subgroups, AI and AII, were significantly different from B. byssoidea (P < 0.001), and that B. aclada subgroups AI and AII were significantly different from each other (P < 0.001). No UP-PCR alleles were shared by AI and B. byssoidea isolates, whereas 10 and 12 alleles were shared by AI:AII and AII:B. byssoidea, respectively. The hypothesis that AII may be a hybrid between AI and B. byssoidea is discussed.

Analysis of two incompletely spliced Arabidopsis cDNAs encoding novel types of peroxidase.

Most known class III peroxidase genes contain three introns at conserved positions. Two Arabidopsis cDNAs (ESTs), encoding novel type peroxidases ATP9a and ATP15a were sequenced, and found to contain inserts for intron 2. PCR and sequence analysis of genomic DNA revealed that the atp9a gene contains all three introns, whereas atp15a contains only introns 2 and 3. The ATP15a cDNA intron contained a single base substitution reducing the splicing potential significantly as compared with the genomic sequence. The putative enzymes share essential catalytic and structural features with horseradish peroxidase, despite a pair-wise sequence identity of only 40-45% among the three.

Disparate sequence characteristics of the Erysiphe graminis f.sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene.

The Erysiphe graminis f.sp. hordei (Egh) glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated and characterized. It contains typical promoter elements and has three introns, one of which is positioned in the 5' untranslated region of the gene. The deduced amino-acid sequence has 87% similarity to gpd genes from other Ascomycete fungi. This is at the same level as previously estimated among these fungi. Comparison at the DNA level reveal similarities of only around 70%, which is 10% lower than previously reported. In an evolutionary tree based on the sequences from 18 fungal gpd genes, Egh falls into the group of Ascomycetes located at a basal position. The regulatory region of the Egh gpd gene has no homology to corresponding sequences in other filamentous Ascomycetes. Codon usage was determined for the four characterized Egh genes (tub2, Egh7, Egh16 and gpd) and found to be similar for all four genes. The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position. Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal and plant genes in sequence mixtures. The Egh gpd promoter appears to be superior to that of the Egh beta-tubulin gene (tub2) for driving the E. coli beta-glucuronidase (GUS) gene in transformation experiments.