Edgar Schliephake

Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate

Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance.

Barley yellow dwarf virus transmission and feeding behaviour of Rhopalosiphum padi on Hordeum bulbosum clones

A Hordeum bulbosum L. (Poaceae) clone A17 was identified, which showed complete resistance to Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV). It was not possible to infect plants of A17 with BYDV‐PAV, ‐MAV, or with CYDV‐RPV by the aphid vectors Rhopalosiphum padi (L.) or Sitobion avenae (Fabricius) (both Hemiptera: Aphididae). Plants of the A17 clone and of the BYDV‐susceptible H. bulbosum clone A21 revealed some resistance to R. padi compared to the susceptible winter barley cultivar Rubina [Hordeum vulgare L. (Poaceae)]. The development time to the imago was longer and the number of nymphs was reduced on both clones compared with cv. Rubina. The probing and feeding behaviour of R. padi on plants of the H. bulbosum clones was studied over 12 h and compared with that on plants of the barley cv. Rubina. Principal component analysis of the results of the feeding behaviour revealed a clear separation of the H. bulbosum genotypes from Rubina. On H. bulbosum the number of penetrations was higher but total feeding time was shorter. Significant differences were mainly found in the phloem feeding parameters for plants of both clones in comparison to Rubina, with the virus resistant A17 clone having the strongest effect and the susceptible A21 clone being intermediate. Most significant differences were found in parameters of the phloem salivation phase. On A17, an average of less than one (0.9) E1 phase per plant was observed (3.3 on A21 and 5.7 on Rubina) and its duration was reduced to less than 1 min (0.9 min) in comparison to 2.4 min on A21 and 5.7 min on Rubina. Also, the phloem feeding (E2) phase was clearly reduced on A17 plants with 0.5 E2 phases per test and a mean duration of 1.1 min in contrast with 2.9 and 3.5 E2 phases per test and 34.1 and 421.3 min for A21 and Rubina, respectively. These results point towards a phloem‐localized factor for aphid resistance in H. bulbosum, i.e., on A17 plants the phloem salivation time is too short for a successful infection by BYDV leading to vector resistance.

Feeding behavior of aphids on narrow-leafed lupin (Lupinus angustifolius) genotypes varying in the content of quinolizidine alkaloids

Since the beginning of breeding narrow‐leafed lupins [Lupinus angustifolius L. (Fabaceae)] with a low alkaloid content, susceptibility to several aphid species has increased. Therefore, the probing and feeding behavior of Aphis fabae Scopoli, Aphis craccivora Koch, Acyrthosiphon pisum (Harris), Myzus persicae (Sulzer), and the well‐adapted Macrosiphum albifrons Essig (all Hemiptera: Aphididae) was studied over 12 h on narrow‐leafed lupin genotypes containing varying amounts and compositions of alkaloids. We used the electrical penetration graph (EPG) technique to obtain information on the influence of alkaloid content and composition on the susceptibility to various aphid species. Results indicated that the total time of probing of A. fabae, A. craccivora, A. pisum, and M. persicae increased with a reduced alkaloid content, whereas the alkaloid content had no influence on M. albifrons. Almost all of the individuals (>93%) conducted sieve element phases on the highly susceptible genotype Bo083521AR (low alkaloid content). A reduced occurrence of phloem phases was observed during the 12‐h recording on the alkaloid‐rich cultivar Azuro, especially for A. pisum (37.5%) and A. fabae (55.0%). Furthermore, aphids feeding on genotypes with low alkaloid content had in most cases significantly longer sieve element phases than when feeding on resistant genotypes (Kalya: low alkaloid content, yet resistant; Azuro: high alkaloid content, resistant), whereas M. albifrons showed the longest phloem phase on the alkaloid‐rich cultivar Azuro. As most significant differences were found in phloem‐related parameters, it is likely that the most important plant factors influencing aphid probing and feeding behavior are localized in the sieve elements. The aphids’ feeding behavior on the cultivar Kalya, with a low alkaloid content but reduced susceptibility, indicates that not only the total alkaloid content influences the feeding behavior but additional plant factors have an impact.

Diagnostic value of molecular markers for Lr genes and characterization of leaf rust resistance of German winter wheat cultivars with regard to the stability of vertical resistance

Breeding for resistance is an efficient strategy to manage wheat leaf rust caused by Puccinia triticina f. sp. tritici. However, a prerequisite for the directed use of Lr genes in breeding and the detection of new races virulent to these Lr genes is a detailed knowledge on Lr genes present in wheat cultivars. Therefore, respective molecular markers for 18 Lr genes were tested for specificity and used to determine Lr genes in 115 wheat cultivars. Results obtained were compared to available pedigree data. Using respective molecular markers, genes Lr1, Lr10, Lr26, Lr34 and Lr37 were detected, but data were not always in accordance with pedigree data. However, leaf rust scoring data of field trials confirmed the reliability of DNA markers. These reliable marker data facilitated the analyses of the development of virulent leaf rust races from 2002 to 2009 based on released cultivars. A sudden change from low infection rates to susceptibility was observed for Lr1, Lr3, Lr10, Lr13, Lr14, Lr16, Lr26 and Lr37 since 2006. Cultivars carrying several leaf rust resistance genes showed no significant shift to susceptibility except one cultivar which revealed an increasing infection rate at a low level. In summary, it turned out that pedigree data are often not reliable and a detection of Lr genes by diagnostic markers is fundamental to combine Lr genes in cultivars for a durable resistance against leaf rust, and to conduct reliable surveys based on released cultivars, instead of ‘Thatcher’ NILs.

Effect of the Soil pH on the Alkaloid Content of Lupinus angustifolius

Field studies were conducted in growing seasons 2004, 2005, and 2010 to investigate the effect of different soil pH values on the alkaloid content in seeds of Lupinus angustifolius. Two-year experiments with eleven cultivars were carried out in acid soils with an average of (Mecklenburg-Western Pomerania) and on calcareous soils with an average pH of 7.1 (Bavaria), respectively. In addition, in 2010, eight cultivars were grown in field experiments in soils with pH values varying between and . In all experiments conducted on soils with a higher pH ( and ), a significantly lower alkaloid content was detected in all Lupinus angustifolius cultivars than on soils with a lower pH ( and ). Results clearly show that the alkaloid content is significantly influenced by the soil pH but genotypic differences regarding the reaction to different pH values in the soil were observed.

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1

Key messageSNP markers were developed for the OWBM resistance geneSm1that will be useful for MAS. The wheatSm1region is collinear with an inverted syntenic interval inB. distachyon.AbstractOrange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Penetration behavior of different aphid species on Lupinus angustifolius L. genotypes

The main role of MADS-box transcription factors in plant developmental processes has been well described in the model plant Arabidopsis thaliana. However, little is known about their function in crops of important agricultural and commercial value. Our study aims to investigate their role in two agronomical relevant Rosaceae crops: apple (Malus x domestica Borkh.) and strawberry (Fragaria vesca). Expression studies using qPCR and RNA seq have identified two apple Dormancy Associated MADS-box (DAM) genes. They group with the StMADS11 clade, and were named MdDAM1 and MdDAM2, the last one discovered ex novo. Real time expression studies in dormant buds collected during the chilling period and chromatin immunoprecipitation (ChIP) analyses confirmed that the genes are downregulated by exposure to cold and MdDAM1 is epigenetically repressed, as it has been demonstrated for Arabidopsis FLC and peach DAM genes. In parallel we worked on strawberry MADS-box genes of known function involved in flower development. We chose three MADS-box genes that are homologs of Arabidopsis PISTILLATA and AGAMOUS to perform gene expression and functional analysis using a RNA interference approach to obtain post-transcriptional gene silencing. The positive transgenic lines of each transformation were evaluated at the molecular and phenotypic level. Single gene mutants does not show altered flower phenotype, suggesting a different mechanism of flower development in strawberry, probably due to the peculiar flower structure.The quarantine pest pine wood nematode (PWN) Bursaphelenchus xylophilus, native in North America, can be found in wood and root parts of infected conifer trees with focus on Pinus species. At the beginning of the 20 century this species was introduced into Japan supposedly with timber imports where it induced pine wilt expression on susceptible native pine species. Further outbreaks outside Europe are reported from China, Korea, Taiwan and Mexico. In 1999 PWN was found in Portugal, which today is declared as complete infested, since followed by first single tree outbreaks in Spain. The European Union (EU) member states have to undertake measures to prevent further spread of this nematode according to the emergency measures of the EU Commission.The box tree pyralid Cydalima perspectalis (Walker 1859) is an invasive alien moth from East Asia which occurs in Central Europe since 2007. It is an insect pest on plants of the genus Buxus, causing serious damage. Because of the rapid spread in Germany and nearby countries like Switzerland, one part of this study was testing different ways of eco-friendly regulation with commercially available beneficials and biological control agents.1 Julius Kühn-Institute, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany. 2 Geisenheim Research Center, Section Phytomedicine, Geisenheim, Germany. 3 Área de Genética, Facultad de Ciencias, Universidad de Málaga, Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC), Málaga (Spain). 4 Suez Canal University, Faculty of Agriculture, Ismailia, Egypt. Email of corresponding author: namis.eltlbany@jki.bund.deExcessive use of mineral nitrogen (N) fertilizer is a common practice in rice-wheat rotations in southeastern China. At the same time N use efficiencies (NUEs) in these rice-based cropping system are very low (Peng et al., 2006). The consequences are high N losses from arable land to water bodies (surfaceand groundwater) and to the atmosphere (Richter and Roelcke, 2000). To investigate the scope and scale of reductions in mineral N fertilizer inputs, demonstration field experiments on farmers’ field sites were conducted for three consecutive winter wheat-summer rice double crop rotations in two counties of Jiangsu Province from 2008 to 2011. Results from the first two years are presented here. NUEsPolycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants which often persist in soil. The biochemistry of microbe-driven degradation of PAHs is well-studied though less is known about abiotic soil environmental factors influencing the microbe-phenanthrene interaction. Since minerals and charcoal are major soil components we matured different microbial communities in artificial soils based only on variation in the mineral content (montmorillonite, illite, ferrihydrite) and presence of charcoal for 2 years after addition of Luvisol microorganisms and sterile manure. Established microbial communities in soils were exposed to PAHs’ model compound phenanthrene (2 mg/g) to study microbial functionality depending on soil composition. Furthermore, litter which was previously shown to enhance microbial activity was added to the soils (1 wt%). Both treatments with corresponding controls were further incubated for 63 days with sampling on day 0, 7, 21 and 63. A natural Luvisol soil was treated similarly for control. Fingerprints by denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA or ITS gene fragments, respectively, amplified from extracted total community DNA were generated. The presence of phenanthrene-degradative genes was screened by PCR-Southern Blot detection and soil treatments were subjected to a chemical phenanthrene analysis. DGGE fingerprints revealed that the addition of phenanthrene and litter caused a shift in microbial community composition. Differences in bacterial response to phenanthrene were seen depending on litter addition and soil composition. The incubation time was determined as additional influencing factor. Furthermore, a dominance of a specific genotype for phenanthrene degradation is assumed based on PCR-Southern Blot analysis.1 Julius Kühn-Institute Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11-12, D-38104 Braunschweig 2 Institute of Crop Science and Resource Conservation, Soil Science and Ecology, University of Bonn, Nussallee 13, D-53115 Bonn 3 Institute of Bioand Geosciences 3, Agrosphere, Forschungszentrum Jülich GmbH, D-52425 Jülich Email of corresponding author: sven.jechalke@jki.bund.de

Chromosomal assignment of oil radish resistance to Meloidogyne incognita and M. javanica using a set of disomic rapeseed-radish chromosome addition lines

Root-knot nematodes cause severe damage to a great number of crops worldwide. The use of nematicides is restricted due to environmental and toxicological risks and control of the pest by crop rotation is difficult because root-knot nematodes have a very wide range of host plants. To verify the strategy of converting rapeseed from a tolerant host for Meloidogyne incognita and M. javanica to a resistant catch crop, a complete set of nine disomic rapeseed-radish chromosome addition lines (lines A to I) was tested for resistance against these Meloidogyne species. Thirty plants of each addition line and the rapeseed and radish parents as control were infected with 2500 second-stage juveniles per plant. The presence of the alien radish chromosome was confirmed by chromosome-specific microsatellite markers. After cultivation of the inoculated plants for 10 weeks in a climatic chamber the root systems were washed. The egg masses were stained with Cochenille Red and counted. The radish parent A24 was found to be resistant to M. incognita (2.4 egg masses (g root)−1) and M. javanica (0.4 egg masses (g root)−1) compared to 53.3 and 33.1 egg masses (g root)−1 for the susceptible rapeseed parent cv. Madora. The radish chromosome e was shown to be the carrier of radish root-knot nematode resistance with an average number of <1 egg mass (g root)−1 for M. incognita and M. javanica. The disomic addition lines B, C, D, G, H and I and the parental radish line A107 were classified as highly susceptible, whereas the addition lines A and F showed significantly reduced susceptibility for M. incognita but not for M. javanica. To our knowledge this is the first study on resistance effects of individual radish chromosomes in a rapeseed background against these root-knot nematodes.

Association genetics studies on frost tolerance in wheat ( Triticum aestivum L.) reveal new highly conserved amino acid substitutions in CBF-A3 , CBF-A15 , VRN3 and PPD1 genes

BackgroundUnderstanding the genetic basis of frost tolerance (FT) in wheat (Triticum aestivum L.) is essential for preventing yield losses caused by frost due to cellular damage, dehydration and reduced metabolism. FT is a complex trait regulated by a number of genes and several gene families. Availability of the wheat genomic sequence opens new opportunities for exploring candidate genes diversity for FT. Therefore, the objectives of this study were to identity SNPs and insertion-deletion (indels) in genes known to be involved in frost tolerance and to perform association genetics analysis of respective SNPs and indels on FT.ResultsHere we report on the sequence analysis of 19 candidate genes for FT in wheat assembled using the Chinese Spring IWGSC RefSeq v1.0. Out of these, the tandem duplicated C-repeat binding factors (CBF), i.e. CBF-A3, CBF-A5, CBF-A10, CBF-A13, CBF-A14, CBF-A15, CBF-A18, the vernalisation response gene VRN-A1, VRN-B3, the photoperiod response genes PPD-B1 and PPD-D1 revealed association to FT in 235 wheat cultivars. Within six genes (CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1) amino acid (AA) substitutions in important protein domains were identified. The amino acid substitution effect in VRN-A1 on FT was confirmed and new AA substitutions in CBF-A3, CBF-A15, VRN-B3, PPD-B1 and PPD-D1 located at highly conserved sites were detected. Since these results rely on phenotypic data obtained at five locations in 2 years, detection of significant associations of FT to AA changes in CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1 may be exploited in marker assisted breeding for frost tolerance in winter wheat.ConclusionsA set of 65 primer pairs for the genes mentioned above from a previous study was BLASTed against the IWGSC RefSeq resulting in the identification of 39 primer combinations covering the full length of 19 genes. This work demonstrates the usefulness of the IWGSC RefSeq in specific primer development for highly conserved gene families in hexaploid wheat and, that a candidate gene association genetics approach based on the sequence data is an efficient tool to identify new alleles of genes important for the response to abiotic stress in wheat.

Evaluation of genetic resources in the genus Asparagus for resistance to Asparagus virus 1 (AV-1)

Forty-four Asparagus officinalis cultivars, gene bank accessions and breeding lines as well as thirty-four accessions of wild relatives of Asparagus were evaluated for resistance to Asparagus virus 1. Three different test strategies were developed for the assessment of individual plants: (1) natural infection under field conditions, or two vector-mediated infection assays using the green peach aphid Myzus persicae (2) in an insect-proof gauze cage or (3) in a climate chamber. The AV-1 infections were verified by DAS-ELISA and RT-PCR approaches. All tested 660 individual plants of A. officinalis germplasm were susceptible to AV-1 infection. In contrast, in 276 plants of 29 Asparagus wild accessions no virus infection could be detected. These resistant accessions comprised of nineteen diploid, tetraploid and hexaploid species of both the Eurasian clade and the African clade of the asparagus germplasm. Data of the AV-1 resistance evaluation are discussed in relation to the genetic distance of the resistance carrier and potential application in breeding.