Confirming infection of hop plants inoculated with Verticilium nonalfalfae.

Hop (Humulus lupulus L.) is grown mostly as flavouring and bittering ingredient for beer and is also appreciated in the herbal and cosmetic industry, as well as in pharmacology. Among several diseases that damage hop growing, the most devastating in European hop production is verticillium wilt, caused by the soil-borne fungal pathogen Verticillium nonalfalfae. Colonization pattern and differential expression of selected genes after artificial infection of susceptible and resistant hop cultivars with V. nonalfalafae in stems and roots have been analysed recently Svara et al., 2019. Here, we present the dataset related to verification of plant samples infections after artificial inoculation (fungi- and mock-inoculated). After inoculation plant samples were tested for the positive infection by PCR amplification of the V. nonalfalfae ITS DNA region with species specific primers developed and optimised for this purpose. For more insight please see the article "Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae".

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.

Propagation and some physiological effects of Citrus bark cracking viroid and Apple fruit crinkle viroid in multiple infected hop (Humulus lupulus L.).

The hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple viroid infections affecting hop physiology. Combinatorial biolistic hop inoculation with Citrus bark cracking viroid (CBCVd), Apple fruit crinkle viroid (AFCVd), Hop latent viroid, and Hop stunt viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other viroid combinations were highly compatible. Unlike to other viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of hop with all viroids led to multiple infections with unstable viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.

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.

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.