Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae.

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

VALIDATING FORWARD MODELING AND INVERSIONS OF HELIOSEISMIC HOLOGRAPHY MEASUREMENTS.

Here we use synthetic data to explore the performance of forward models and inverse methods for helioseismic holography. Specifically, this work presents the first comprehensive test of inverse modeling for flows using lateral-vantage (deep-focus) holography. We derive sensitivity functions in the Born approximation. We then use these sensitivity functions in a series of forward models and inversions of flows from a publicly available magnetohydrodynamic quiet-Sun simulation. The forward travel times computed using the kernels generally compare favorably with measurements obtained by applying holography, in a lateral-vantage configuration, on a 15-hour time series of artificial Dopplergrams extracted from the simulation. Inversions for the horizontal flow components are able to reproduce the flows in the upper 3Mm of the domain, but are compromised by noise at greater depths.

First Report of Downy Mildew Caused by Hyaloperonospora camelinae on Camelina sativa in Slovenia.

Camelina or false flax (Camelina sativa), of the Brassicaceae, is an annual flowering plant native to Europe and Central Asia where it is grown commercially as an oilseed crop. At the end of May 2012, symptoms of downy mildew were observed on camelina plants grown in the Savinja Valley in Slovenia. The disease was found in four monitored fields (total area 3 ha), and the incidence ranged from 2 to 38% depending on the variety. Symptomatic plants showed whitish, abundant, and fluffy mycelia covering the stems, flowers, seed pods, and undersides of the leaves. The disease mainly affected the upper half of the plants, and the stems were reduced and distorted. During disease progression, the mycelium turned from gray to black. Microscopic observations revealed hyaline, straight conidiophores that were branched monopodially (3 to 4 times) with 6 to 12 re-curved tips/branch, and measured 140 to 300 × 12 to 20 µm. Conidia were hyaline, oval to broadly ellipsoidal, 24 to 29 × 18 to 24 µm. Oospores formed in necrotic stem and leaf tissues were dark brown and measured 30 to 38 µm in diameter. Based on these morphological characteristics, the causal agent was identified as Hyaloperonospora camelinae (1,3,4,5). DNA was extracted from mycelium and conidia collected from infected plants in two fields in the Savinja Valley (1HpC and 2HpC). Nuclear internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified by PCR assay from two isolates using the universal primers ITS4 and ITS5, and sequenced. Both samples yielded a 781-bp sequence, which showed 100% identity to H. camelinae ITS sequence JX445136 in GenBank. The nucleotide sequence was assigned to GenBank Accession No. KJ768405. Pathogenicity was confirmed by spraying 25 3-week-old plants of C. sativa cv. Ligena planted in pots (5 plants/pot) with a conidial suspension (105 conidia/ml) obtained from 10 infected plants of the same variety collected from the field 1HpC. Inoculated plants were covered with polyethylene bags for 2 days to maintain high humidity, and incubated at 20°C with a 12-h photoperiod/day in a growth chamber. Downy mildew symptoms first developed on leaves 6 days after inoculation. An additional 25 control plants sprayed with sterilized distilled water and otherwise treated similarly to the inoculated plants developed no symptoms. The identity of the pathogen on the inoculated plants as H. camelinae was confirmed based on the morphological features described above. Downy mildew of false flax caused by H. camelinae has been reported in Europe from Austria, Bulgaria, Germany, Poland, Portugal, Spain, and Switzerland (2); and in the United States from Florida, Oregon, Minnesota, Montana, Nebraska, and Washington (1,3,4,5). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Slovenia. The representative samples were deposited in the phytopatological herbarium of the Slovenian Institute of Hop Research and Brewing. References: (1) E. M. Babiker et al. Plant Dis. 96:1670, 2012. (2) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ . (3) R. M. Harveson et al. Plant Health Progress. doi: 10.1094/PHP-2011-1014-01-BR, 2011. (4) M. L. Putnam et al. Plant Health Progress. doi: 10.1094/PHP-2009-0910-01-BR, 2009. (5) P. Srivastava et al. Plant Dis. 96:1692, 2012.

First Report of Powdery Mildew Caused by Golovinomyces biocellatus on Common Sage (Salvia officinalis) in Slovenia.

Common sage (Salvia officinalis) is a well known perennial and medicinal herb in the Lamiaceae family, which is widely planted in gardens and parks in Slovenia. In September 2007, symptoms of powdery mildew infection were observed on common sage plants grown in several gardens in the Savinja valley. White mycelium was present, principally on the upper leaf surface, but was also observed on stems. The disease progressed as spots coalesced and leaves become distorted and necrotic. Microscopic observations revealed septate and branched hyaline hyphae 4 to 7 µm wide. Conidiophores were cylindrical and septate and measured 40 to 90 × 9 to 12 µm. The foot cells of the conidiophores were straight, followed by one to three shorter cells. Conidia produced in chains (three to four conidia per chain) were hyaline and doliform in shape, measuring 27 to 35 × 14 to 20 µm and lacking fibrosin bodies. Cleistothecia were not observed in the collected samples. All of these characteristics were consistent with Golovinomyces biocellatus as described by Braun (2). For molecular identification of the pathogen, DNA was extracted from mycelia and conidia of infected plants, collected in two different gardens in the Savinja valley as representative samples (1GB-Sof and 2GB-Sof). Nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR using the universal primers ITS4 and ITS5, and sequenced. Both samples yielded the same 532 bp sequence, which showed the highest identity (97 to 99%; E value = 0.0) to G. biocellatus ITS sequences in the NCBI GenBank (1). The nucleotide sequence has been assigned GenBank Accession No. JQ340358. Pathogenicity was confirmed by inoculation of 10 healthy plants of S. officinalis 'Grower's Friend' planted in pots. Plants were sprayed with a spore suspension (105 conidia/ml; 0.01% Tween 20) obtained from naturally infected leaves. Inoculated plants were covered with polyethylene bags for two days to maintain high humidity and incubated in a growing chamber at 22°C with a 12-h photoperiod. The first powdery mildew signs and symptoms developed on leaves 7 days after inoculation. Ten control plants sprayed with distilled water showed no symptoms. The fungus present on the inoculated plants was morphologically identical to that originally observed on diseased plants. Powdery mildew infections of common sage associated with G. biocellatus have been known in Argentina, Washington State (United States), and various countries in Europe (2,3,4). To the best of our knowledge, this is the first report of G. biocellatus on common sage in Slovenia. Voucher specimens are available at the culture collection of the Slovenian Institute of Hop Research and Brewing. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (3) M. G. Cabrera et al. Mycosphere 1:289, 2010. (4) F. M. Dugan. North American Fungi 6:1, 2011.

First Report of Hop stunt viroid Infecting Hop in Slovenia.

Hop (Humulus lupulus), of the Cannabaceae family, is a dioecious perennial climbing plant that is native to Asia, North America, and Europe and is commercially grown in many countries for its use in brewing and the pharmaceutical industry. Slovenia has a more than 100-year-old hop-growing tradition and it is an important national agricultural business, with 90% of production exported to foreign markets. Since 2007, symptoms similar to Hop stunt viroid (HSVd) infection have been observed in several hop gardens with cvs. Celeia, Bobek, and Aurora in the Savinja Valley and Koroska Region. Symptoms include stunting, leaf curl, small cone formation, and dry root rot. In the first year of finding the disease, the incidence varied from 1 to 30% and increased rapidly (by as much as 10%) each subsequent year, predominantly along plant rows. For molecular identification of the pathogen, RNA was extracted from leaves and cones of symptomatic and asymptomatic plants from two different hop gardens with cv. Celeia using Tri Reagent (T9424; Sigma-Aldrich, St Louis, MO). Reverse transcription-PCR was carried out using two pairs of specific HSVd primers, HSVdI/HSVdII and HSVdeI/HSVdeII (3,4). Both primer pairs gave a single PCR product from tissue from symptomatic plants, with expected lengths of ~300 bp, but no amplicons were produced using samples from asymptomatic plants. PCR products from HSVdI/HSVdII were subjected to direct sequencing and HSVdeI/HSVdeII products were cloned in PCR Script SK (+) (Stratagene, La Jolla, CA) vector and sequenced. Five sequences (EMBL Accession Nos. HE575344, HE575345, HE575346, HE575347, and HE575348) were obtained, which revealed 96 to 99% sequence identity with various HSVd variants (grapevine, citrus, and cucumber) reported in GenBank of the National Centre for Biotechnology Information (NCBI). HSVd belonging to the Hostuviroid genus, Pospiviroidae family, has been previously reported in hop in Japan, South Korea, North America, and China (1,2). To our knowledge, this is the first report of the detection of HSVd on hop in Europe. Strict phytosanitary measures have been taken to prevent further spread and to eradicate HSVd infections. References: (1) K. C. Eastwell and T. Sano. Hop Stunt. Page 48 in: Compendium of Hop Diseases and Pests. W. F. Mahaffee et al., eds. The American Phytopathological Society, St. Paul, MN, 2009. (2) L. Guo et al. Plant Pathol. 57:764, 2008. (3) J. Matousek et al. Plant Soil Environ. 49:168, 2003. (4) J. Matousek et al. J. Virol. Methods 122:153, 2004.

High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT).

Implementation of molecular methods in hop (Humulus lupulus L.) breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. However, use of molecular marker technology is limited due to expense, time inefficiency, laborious methodology and dependence on DNA sequence information. Diversity arrays technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of quality polymorphic markers without reliance on DNA sequence information. This study is the first to utilise DArT for hop genotyping, identifying 730 polymorphic markers from 92 hop accessions. The marker quality was high and similar to the quality of DArT markers previously generated for other species; although percentage polymorphism and polymorphism information content (PIC) were lower than in previous studies deploying other marker systems in hop. Genetic relationships in hop illustrated by DArT in this study coincide with knowledge generated using alternate methods. Several statistical analyses separated the hop accessions into genetically differentiated North American and European groupings, with hybrids between the two groups clearly distinguishable. Levels of genetic diversity were similar in the North American and European groups, but higher in the hybrid group. The markers produced from this time and cost-efficient genotyping tool will be a valuable resource for numerous applications in hop breeding and genetics studies, such as mapping, marker-assisted selection, genetic identity testing, guidance in the maintenance of genetic diversity and the directed breeding of superior cultivars.

New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L.

One hundred and thirty-five microsatellite markers were developed for hop Humulus lupulus L. from di- and trinucleotide-enriched libraries. Seventy-eight primers showed amplification in two tested genotypes. Twenty-four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop.

Xiphinema rivesi from Slovania Transmit Tobacco ringspot virus and Tomato ringspot virus to Cucumber Bait Plants.

The dagger nematode, Xiphinema rivesi Dalmasso, a member of the X. americanum group, was detected in 2002 for the first time in Slovenia and for the fourth time in Europe (4). X. rivesi is a vector of at least four North American nepoviruses including Cherry rasp leaf virus (CRLV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Peach rosette mosaic virus (PRMV) (1,2). All of these viruses are included on the EPPO and EU lists of quarantine organisms, but none of the Xiphinema species found in Europe have been reported to transmit these nepoviruses. Three virus isolates, including TRSV (from Lobelia spp.; virus collection of the Plant Protection Service, Wageningen, The Netherlands), ToRSV (grapevine isolate PV-0381; DSMZ, Braunschweig, Germany), and Arabis mosaic virus (ArMV) (from Vinca spp.; virus collection of the Plant Protection Service), were used in transmission tests with a population of X. rivesi found in Slovenia. X. rivesi is not known to transmit ArMV and this virus was included as a check. The nematodes were extracted from peach orchard soil collected near the village of Dornberk, and transmission tests fulfilled the set of criteria proposed by Trudgill et al. (3). Cucumis sativus cv. Eva, grown in a growth chamber at 25°C, was used as acquisition hosts and transmission bait plants. The acquisition hosts were mechanically inoculated and showing systemic symptoms before the introduction of nematodes. Noninoculated acquisition plants were included as controls. After a 10-day acquisition feeding period, the nematodes were transferred to healthy bait plants and allowed a 14-day inoculation feeding period. X. rivesi transmitted TRSV and ToRSV but not ArMV. TRSV and ToRSV bait plants developed systemic symptoms 4 to 6 weeks after the nematodes were transferred. Transmission of TRSV and ToRSV was confirmed by testing leaf and root sap of bait plants in a double antibody sandwich (DAS)-ELISA. High virus concentrations were detected in the roots and leaves of TRSV and ToRSV symptomatic plants. DAS-ELISA on bait plants from nematodes that had been allowed to feed on ArMV-infected or the virus-free control acquisition plants gave negative results. No symptoms appeared on bait plants used for ArMV transmission or the control bait plants. To our knowledge, this is the first report of transmission of TRSV and ToRSV with a Xiphinema population from Europe. References: (1) D. J. F. Brown et al. Phytopathology 84:646, 1994. (2) L.W. Stobbs et al. Plant Dis. 80:105, 1996. (3) D. L. Trudgill et al. Rev. Nematol. 6:133, 1983. (4) G. Urek et al. Plant Dis. 87:100, 2002.

Molecular phylogeny of wild hops, Humulus lupulus L.

We have analysed wild hops collected widely from the Northern Hemisphere, assessing the genetic diversity and the geographical distribution of haplotypes, to investigate the evolution and phylogeny of hops, Humulus lupulus. The haplotypes were characterized by the nuclear ribosomal DNA spacer region (length and DNA sequence) and chloroplast DNA noncoding regions (DNA sequences). The results indicated that primary divergence into European (including Caucasus and Altai hops), and Asian-North American types, was 1.05+/-0.28 to 1.27+/-0.30 million years ago. Although an Eastern boundary for European nuclear haplotype distribution was unclear due to the ambiguous origin of Northern Chinese samples, the European hop group showed a wide geographical distribution across Eurasia from the Altai region to Portugal. The low genetic variation in this group suggested rapid and recent expansion. The North American hop group showed high diversity, and is considered to include hops that have migrated from Asia. Japanese and Chinese hops were identified as genetically distinct. This study has shown that wild hops in each growing region are genetically differentiated with considerable genetic diversity. It gives insights into the evolution and domestication of hops that are discussed.

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.

Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX.

Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within any organism. Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX. We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes. Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding. We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts. Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.

Random amplified polymorphic DNA analysis, genome size, and genomic in situ hybridization of triploid viviparous onions

Triploid viviparous onions (Allium cepa L. var. viviparum Metzg. (ALEF.), auct.), (2n = 3x = 24), are known in some countries only as a rare relic crop, while in other parts of the world they are still traditionally or even commercially cultivated. Results indicating an identical random amplified polymorphic DNA (RAPD) banding pattern and the same DNA content (2C = 43.4 pg) establish the high genetic similarity and the unique origin of the Croatian clone Ljutika and the Indian clone Pran. In order to determine the parental Allium species of these natural triploid hybrids, genomic fluorescent in situ hybridization (GISH) was applied. Biotinylated genomic DNAs from six diploid Allium species (A. cepa L., A. fistulosum L., A. roylei Stearn, A. vavilovii M. Pop. et Vved., A. galanthum Kar. et Kir., A. oschaninii O. Fedtsch.) were used as probes in this study. While probes obtained from genomic DNA of A. cepa, A. vavilovii, and A. roylei hybridized to somatic chromosomes of Ljutika probes from A. fistulosum, A. galanthum, and A. oschaninii did not. The DNA probes of A. cepa and A. roylei each completely or predominantly labelled one genome (eight chromosomes). A few chromosomes, the markers of the triploid karyotype, were not completely labelled by any probe applied. Our GISH results indicate that triploid viviparous onions might possess a complex triparental genome organization.

Gynogenic lines of onion (Allium cepa L.): evidence of their homozygosity.

Haploid induction via gynogenesis offers the possibility of using doubled haploid (DH) inbred lines in onion breeding. A first DH line that originated from the open-pollinated (OP) cultivar 'Dorata di Parma' was obtained after overcoming difficulties associated with the haploidy of the regenerants. Spontaneous chromosome doubling occurs seldom in onion. The first DH line obtained was cloned and selfed to produce sufficient seeds for genetic studies. The homozygosity of the DH gynogenic line was revealed on the basis of the low standard deviations of the bulb traits polar diameter, shape index and weight with respect to those of the S1 line or the OP cultivar. In the DH line, moreover, segregation of RAPD and alpha esterase markers was not noted. Out of four primers revealing polymorphism at 16 ge-netic loci in the OP cultivar 'Dorata di Parma', none produced polymorphism in the DH gynogenic line. The Est-1 locus, homozygous in 22 plants (Est-1 (1/1) in 3 and Est-1 (2/2) in 19) and heterozygous (Est-1 (1/2)) in 11 plants of the OP cultivar, always carried the same alleles in the DH line. We also tested genetic stability during micropropagation of a second halpoid line obtained via gynogenesis from var. 'Senshyu Yellow'. Seventeen plants of this line were tested to detect changes occurring during the tissue culture process. Again no polymorphism was observed. The high genetic homogeneity observed in the two gynogenic lines of onion could be related to the absence of the callus phase during the gynogenic process.