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Grape hyacinths (Muscari spp.) are popular spring flowering bulbs in Europe and also in Hungary. In the spring of 2017, we came across grape hyacinth plants showing mosaic symptoms, which indicated viral infection. Currently Hyacinth mosaic virus (genus Potyvirus), a proposed member of the genus named Muscari mosaic virus, Arabis mosaic virus (genus Nepovirus), Cucumber mosaic virus (genus Cucumovirus) and Tobacco rattle virus (genus Tobravirus) are known to infect grape hyacinth. Leaf samples of symptomatic grape hyacinths were observed, collected and the presence of potyviruses was proved with potyvirus specific monoclonal antibody by ELISA and by potyvirus specific RT-PCR. Laboratory host plants and seed grown Muscari plants were inoculated with leaf sap and symptoms were recorded. Nucleotide sequences of the cloned fragments were compared to GenBank data. In the case of Muscari ‘Helena’ the highest nucleotide identity in the coat protein coding region was found with two Muscari mosaic virus isolates (95.51% and 95.79%). In the case of the clones derived from Muscari ‘Pink Sunrise’ plant, the highest identity was recorded with Muscari mosaic virus isolates (57.65% and 57.80%) and with a Tulip breaking virus strain (55.13%) indicating the existence of a novel potyvirus species, tentatively named Muscari chlorotic mottle virus. The coat protein sequences were aligned and Maximum Likelihood trees were built to analyze phylogenetic relationships.

WOS: 000504829400003 The tulip (Tulipa sp.) is one of the most important ornamental bulbous plants, which has been cultivated as a cut-flower, potted, and garden plant, and used for landscaping in Turkey. This study investigated the occurrence of a viral disease in the tulip cultivars Strong Gold, Pretty Woman and Purple Prince that causes striping of the leaves, flames of different colours on the petals and mosaic patterns on the leaves, in Samsun province of Turkey. Surveys of virus-infected tulip plants were carried out in the Middle Black Sea Region of Turkey in 2015-2016. A total of 212 samples were collected from four locations and checked by biological, serological and molecular methods for the presence of the Tulip breaking virus (TBV). TBV was detected in the leaves and flowers by double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) in the tulip cultivars (15.5%) tested from Samsun province. TBV infection was found at the highest rate in the cultivar Strong Gold (19.7%), followed by Pretty Woman (14.1%) and Purple Prince (12.8%). The presence of TBV in samples was further confirmed by reverse transcription polymerase chain reaction (RT-PCR) assays. This is the first report on TBV naturally infecting tulips in Samsun province, Turkey. Black Sea Agricultural Research Institute We thank the Black Sea Agricultural Research Institute for supporting this research. We would like to thank Sevcan Sari for helping with ELISA tests and Yasemin Izgi Sarac for supplying the tulip cultivars.

Tulip breaking is economically the most important viral disease of modern-day tulip growing. It is characterized by irregular flame and feather-like patterns in the flowers and mosaic on the foliage. Thirty-two leaf samples were collected from cultivated tulip plants showing tulip breaking syndrome from Hungary in 2017 and 2018. Virus identification was performed by serological (ELISA) and molecular (RT-PCR) methods. All samples proved to be infected with a potyvirus and evidence was provided that three potyvirus species could be identified in the samples: Lily mottle virus (LMoV), Tulip breaking virus (TBV) and Rembrandt tulip-breaking virus (ReTBV). Recombination prediction accomplished with Recombination Detection Program (RDP) v4.98 revealed potential intraspecies recombination in the case of TBV and LMoV. Phylogenetic analyses of the coat protein (CP) regions proved the monophyletic origin of these viruses and verified them as three different species according to current International Committee on Taxonomy of Viruses (ICTV) species demarcation criteria. Based on these results, we analyzed taxonomic relations concerning potyviruses associated with tulip breaking syndrome. We propose the elevation of ReTBV to species level, and emergence of two new subgroups in ReTBV.

Tulip crop production in the Netherlands suffers from severe economic losses caused by virus diseases such as the Tulip Breaking Virus (TBV). Infected plants which can spread the disease by aphids must be removed from the field as soon as possible. As the availability of human experts for visual inspection in the field is limited, there is an urgent need for a rapid, automated and objective method of screening. From 2009-2012, we developed an automatic machine-vision-based system, using classical machine-learning algorithms. In 2012, the experiment conducted a tulip field planted at production density of 100 and 125 plants per square meter, resulting in images with overlapping plants. Experiments based on multispectral images resulted in scores that approached results obtained by experienced crop experts. The method, however, needed to be tuned specifically for each of the data trails, and a NIR band was needed for background segmentation. Recent developments in artificial intelligence and specifically in the area of convolutional neural networks, allow the development of more generic solutions for the detection of TBV. In this study, a Faster R-CNN network is applied on part of the data from the 2012 experiment. The outcomes show that the results are almost the same compared to the previous method using only RGB data.

The objective of this work was to describe the occurrence of quarantine Tulip breaking virus (TBV, synonym Lily mottle virus - LMoV) and Lily symptomless virus (LSV), and their respective molecular analyses, to provide data for supporting TBV removal from the Brazilian A1 quarantine pest list, since this virus has spread among the main commercial lily crops in Brazil. The occurrence of these viruses was detected in 12 cultivation areas through multiplex reverse transcription (RT-PCR), using specific primers to genes encoding the respective coat proteins (CP). Eight fragments of 800 nucleotides (nt) obtained from the LMoV-infected lilies and nine fragments of 600 nt from LSV-infected lilies were sequenced. Phylogenetic tree reconstruction showed a robust branch containing the LMoV Brazilian sequences, other LMoV isolates, TBV, and Tulip band breaking virus, suggesting that all are LMoV isolates, although they are clustered into two subgroups. Phylogenetic analysis also showed a robust branch supporting all Brazilian and other LSV sequences, except for an LSV Japanese isolate. Recombination analyses also showed an LMoV recombinant isolate, whereas no recombination events were found among LSV isolates. Lily mottle virus is the prevalent virus in lily crops in Brazil, in single and mixed infections with LSV or Cucumber mosaic virus (CMV).

The viruses infecting tulips have a big influence on the yield and the quality of bulbs and forced flowers. Commercial bulb production is based on clonal propagation, which leads to the accumulation of viruses. Among 22 viruses occurring in tulips, the most common and the most dangerous are Tulip breaking virus, TBV; Tobacco necrosis virus, TNV; Lily symptomless virus, LSV; Cucumber mosaic virus, CMV and Tobacco rattle virus, TRV. The aim of the research was to check which viruses occur most often on Polish tulip plantations. The research was done on two tulip (Tulipa L.) cultivars ‘Strong Gold’ and ‘Leen van der Mark’ grown at 3 farms situated in different parts of Poland (Warsaw Region, Pomerania and Podlasie) during 2006-2007, and then at 2 farms located in Warsaw Region during 2008-2011. Five of the most important viruses infecting tulips (TBV, TNV, LSV, CMV, and TRV) were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in leaves and in bulbs during the period 2006-2010. In the last year of the research two different strains of TRV were detected (TRV-J and TRV-F) and Tulip virus X (TVX) as well. Search for viruses showed that most often TBV virus was detected both in the leaves and the bulbs regardless of the year and plantations. Yellow flowering cultivar ‘Strong Gold’ was infected by viruses more often than in bi-coloured (with red) ‘Leen van der Mark’, because of the difficulties with effective roguing of infected plants due to inconspicuous symptoms of virus infections on yellow coloured flowers. Other viruses were detected sporadically, however increasing occurrence of LSV and TRV was noticed from year to year. In 2011, TVX virus was detected in a few plants of ‘Strong Gold’ and it was the first case of detection of this virus in tulip in Poland.

The tulip (Tulipa L.) is placed in the Liliaceae Juss. family. The major center of origin is situated in Central Asia. Tulips in Lithuania have been known, grown and was favourite flower for a long time. Comprehensive knowledge of ornamental bulb plants appeared at the end of the 18th century. In this study, the assessment of viral diseases of 263 tulip species and cultivars grown in the Botanical Gardens of Vilnius University was carried out. Basing on the research data obtained in the years of 1997-2010. Infected by viral diseases were selected basing on visual inspection of tulips for symptoms (Navalinskienė, Samuitienė, 2006). Identification of agents was carried out at Plant Virus Laboratory of Institute of Botany applying methods of test-plants, electron microscopy, DAS-ELISA. By resistance to Tulip breaking virus, tulip cultivars were arranged into 4 groups: 1) resistant; 2) moderately resistant; 3) non-resistant; 4) excessively non-resistant. In the study, the collection was analyzed according to classification groups. Triumph tulips and Single late tulips dominate. Among the investigated 15 classification groups of tulips grown in the collection of the Botanical Gardens of Vilnius University, Darwin hybrid tulips, Kaufmanniana, Fosteriana, Greigii, varieties and hybrids and other tulip species and their varieties and hybrids are the most resistant to Tulip breaking virus. Most cultivars of the Double early tulips and Parrot tulips were moderately resistant to Tulip breaking virus. Single early tulips, Triumph tulips, Single late tulips, Lily-flowered tulips and Fringed tulips were rather susceptible to viral diseases.

Interspecific hybridization is an important tool in tulip breeding to transfer some important horticultural traits into new cultivars. The diploid (2n=2x=24) F1 interspecific hybrids between T. gesneriana × T. fosteriana (GF) were backcrossed to diploid T. gesneriana cultivar ‘Yellow Flight’ (G). BC1 plants resulting from these crosses were analysed by genomic in situ hybridization (GISH) to estimate their genome constitution and extent and position of intergenomic recombination. All of the BC1 progenies analysed were diploid (2n=2x=24). GISH enabled unequivocally the identification of parental chromosomes as well as the recombinant chromosomes. Because the T. gesneriana parent was used for backcrossing, the number of G genome chromosomes (chromosomes with centormere of T. gesneriana genome) predominated in the BC1 progenies. The total number of T. fosteriana chromosomes in hybrids ranged from four to ten. There were two types of recombinant chromosomes. Those with a centromere of T. fosteriana chromosome with recombinant segment of T. gesneriana (F/G) and vice versa (G/F). Most recombinant chromosomes contained a single crossover. The percentages of T. fosteriana chromatin in BC1 hybrids ranged from 20 to 24%. INTRODUCTION Introgression of valuable horticultural traits into new cultivars can be accomplished by interspecific crosses followed by backcrossing. Introgression breeding has been used to enrich many cultivated crops e.g., wheat (Wang et al., 1996); potato (Tek et al., 2004); Alstroemeria (Kamstra et al., 2004) and Lilium (Barba-Gonzalez, 2005). In Tulipa, the main goal of modern tulip breeding is the introgression of resistance against Tulip Breaking Virus (TBV) into commercially successful T. gesneriana cultivars (van Creij, 1997). Many crosses have been made between resistant to TBV T. fosteriana cultivars and T. gesneriana cultivars (Van Tuyl and Van Creij, 2007). These crosses generated highly resistant genotypes to TBV called Darwin Hybrid tulips (Van Tuyl and Van Creij, 2007). Introgression breeding requires the technique which enables the monitoring of the presence or the absence of alien chromosomes or the recombination between genomes in F1 hybrids and the backcross progenies. Genomic in situ hybridization (GISH) is a cytogenetic technique which enables distinguishing the parental chromosomes in interspecific hybrids (Chase et al., 2003; Marasek et al., 2004) and detecting the recombinant chromosomes (Ramanna et al., 2003; Kamstra et al., 2004; Barba-Gonzalez et al., 2006). In tulip, GISH has been used to determine the genome composition of cultivars of Darwin Hybrid tulips (Marasek et al., 2006; Marasek and Okazaki, 2007) and ‘Purissima’ hybrids (Marasek and Okazaki, 2008). The aim of this experiment is to assess the genome constitution and extent and position of intergenomic recombination in BC1 plants resulting from crossing T. gesneriana cultivars and the fertile F1 interspecific hybrid T. gesneriana × T. fosteriana. a agnieszkamarasek@wp.pl Proc. 23rd Intl. Eucarpia Symp. (Sec. Ornamentals) on “Colourful Breeding and Genetics” Eds.: J.M. van Tuyl and D.P. de Vries Acta Hort. 836, ISHS 2009 106 MATERIALS AND METHODS Plant Materials BC1 hybrids, 061161 series, were derived from crosses between diploid T. gesneriana cultivar ‘Yellow Flight’ (G) and F1 interspecific hybrid T. gesneriana × T. fosteriana (GF), where the latter was used as pollen donor. Capsules were harvested 9 weeks after pollination. After drying at room temperature seeds were kept at 4°C to overcome dormancy. Seed Germination The seeds used for GISH analysis were surface sterilized with H2SO4 (2 min) followed by treatment with 5% hypochlorite solution for 10 min. The seeds were washed in sterile water and placed in Petri dishes on medium containing half strength MS (Murashige and Skoog, 1962), 3% (w/v) sucrose and 0.7% microagar (pH ~5.6). Seed cultures were kept at 4°C in darkness and the germination was observed 30–40 days from starting the culture. Chromosome Preparation Root tips of seedlings were kept in 0.05% colchicine for 3 h at room temperature, fixed in ethanol-glacial acetic acid (3:1) for at least 2 h and then stored at -20°C until required. The root tip meristems were digested in 0.2% (w/v) pectolyase Y23, 0.2% (w/v) cellulase RS, and 0.2% (w/v) cytohelicase in 10 mM acid-sodium citrate buffer (pH ~4.8) at 37°C for about 2 h. The squash preparations were made in a drop of 45% acetic acid and frozen in liquid nitrogen, the cover slips were removed with a razor blade. The preparations were dehydrated in absolute ethanol and air dried. DNA Probe Preparation and Genomic In Situ Hybridization (GISH) Total genomic DNA was extracted from flower buds using the standard cetyltrimethylammonium (CTAB) method (Murray and Thompson, 1980). Sonicated DNA (1–10-kb) of cultivars ‘Princeps’ (T. fosteriana) and ‘Ile de France’ (T. gesneriana) were used as a probe and labelled by nick translation with either Digoxigenin-11-dUTP or Biotin-16-dUTP, in accordance with the manufacturer’s instruction (Roche Diagnostics GmbH, Mannheim, Germany). Autoclaved for 5 min genomic DNA of T. tarda (100–500 bp) was used as blocking DNA. The in situ hybridization procedure was adopted from Marasek and Okazaki, 2008. The hybridization mixture contained 2× SSC, 50% formamide, 10% dextran sulphate, 1% SDS, 150 ng of each target DNA per slide, and blocking DNA (salmon sperm DNA in 50to 80-fold excess of labelled probes and T. tarda DNA in 35to75fold). Chromosome preparations and denatured (75oC for 10 min) hybridization mixture were denatured together (70oC for 5 min) and allowed to hybridize overnight in a humid chamber at 37oC. The post-hybridization washes were carried out for 15 min in 2× SSC at room temperature, followed by washes in 0.1× SSC at 42oC for 30 min. The wash allows DNA sequences with more then 73% homology to the probe to remain hybridized. Digoxigenin labelled DNA was detected with antidigoxigenin-fluorescein rose in sheep (Boehringer, Mannheim, Germany). Biotin labelled DNA was detected with CY-3 conjugated streptavidin (Vector laboratories). Chromosomes were counterstained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) in Vectashield (Vector Laboratories). Photographs were taken using a Canon digital camera attached to an Axiophot microscope with an appropriate filter and then processed using software (Axio Vision 4.2). The length of chromosomes and centromeric index (percentage of short arm length to the total length of chromosome) were determined using freeware application MicroMeasure http://www.colostate.edu/Depts/Biology/MicroMeasure. Chromosomes were positioned within the karyotype based on the decreasing length of the short arm, centromeric index.

Doelstellingen van het Actieplan “Minder virus in Tulp” zijn: Dit project organiseert de opstart en de uitvoering van een actieplan dat leidt tot minder virus in tulp in een economisch rendabele teelt. De aandacht ligt met name bij TBV, TVX, Augustaziek, ArMV en LSV. Uitvoering van het actieplan moet leiden tot enthousiasme en power in de sector waardoor ook nieuwe inspiratie voor innovaties ontstaat. De beoogde modulaire en stapsgewijze aanpak van dit project zorgt voor een duidelijke afbakening van doelstellingen, activiteiten en budget en maakt tussentijdse nadere invulling van activiteiten mogelijk. Dit verslag beschrijft een aantal onderzoeken die hebben plaatsgevonden vanaf de start van dit Actieplan.

Doelstellingen van het Actieplan “Minder virus in Tulp” zijn: Dit project organiseert de opstart en de uitvoering van een actieplan dat leidt tot minder virus in tulp in een economisch rendabele teelt. De aandacht ligt met name bij TBV, TVX, Augustaziek, ArMV en LSV. Uitvoering van het actieplan moet leiden tot enthousiasme en power in de sector waardoor ook nieuwe inspiratie voor innovaties ontstaat. De beoogde modulaire en stapsgewijze aanpak van dit project zorgt voor een duidelijke afbakening van doelstellingen, activiteiten en budget en maakt tussentijdse nadere invulling van activiteiten mogelijk. Dit verslag beschrijft een aantal onderzoeken die hebben plaatsgevonden vanaf de start van dit Actieplan.

Cucumber mosaic virus (CMV), lily symptomless virus (LSV) and lily mottle virus (LMoV) were detected in three lily cultivars by reverse transcription-polymerase chain reaction (RT-PCR). Results were compared with those obtained in an enzyme-linked immunosorbent assay (ELISA). The amplified DNA fragments with expected size and specific bands for each virus were obtained by RT-PCR using degenerate primers. In 18 out of 22 plants tested at least one virus was detected with RT-PCR in the 18 (81.8%) plants compared to 4 (18.2%) plants with ELISA. The present study showed that the RT-PCR is a more sensitive method than ELISA to determine the presence of viruses in Lilium plants of various species.

The inhibitory effect of mineral oil, synthetic pyrethroid insecticide, organophosphorus insecticide and a pyridine azomethine insecticide on the spread of aphid-borne non-persistently transmitted lily symptomless virus (LSV; carlavirus) and lily mottle virus (LMoV; potyvirus) in Lilium was studied. The control effect of spray mixtures of mineral oil (‘Luxan oil H’) and pyrethroid insecticide deltamethrin (‘Decis’) serially reported about in the past was confirmed. The organophosphorus insecticide acephate (‘Ypsilon’) and the pyridine azomethine insecticide pymetrozine (‘Plenum’) applied as single sprays were about half as effective as deltamethrin in the control of virus spread. The effect of acephate and pymetrozine in mixed sprays with mineral oil proved to be similar in control efficacy as sprays of mineral oil plus pyrethroid. The impact of this newly found effect and mode of action of the systemic insecticides as additive to mineral-oil sprays is discussed. INTRODUCTION The control of viruses in Lilium in the Netherlands is mainly concerned with the prevention of spread of non-persistently aphid-transmitted viruses such as lily symptomless virus (LSV; carlavirus) and lily mottle virus (LMoV; potyvirus; known up till 1990s as tulip breaking virus (TBV); Dekker et al., 1993; Derks et al., 1994). Bradley et al. (1962) reported the inhibition of spread of aphid-borne non-persistently transmitted plant viruses by oil to cover the leaf surfaces. Mineral oils have been used in lilies in the Netherlands since the mid 1960s (Asjes, 1976). The synthetic pyrethroid insecticides, which are effective against both acquisition and inoculation in virus transmission, were introduced in the early 1980s (Asjes, 1981; Gibson et al., 1982; Rice et al., 1983). The addition of synthetic pyrethroids to oil allowed the volume of oil to be halved without loss of efficacy. This practice has been used since the early 1990s (Asjes, 1989; 1991; Asjes and Blom-Barnhoorn, 1994; Asjes and Blom-Barnhoorn, 2000). Sprays are applied weekly in May-July to control rapid spread, and fortnightly in August-September to control the slower spread. They are applied at a pressure of 400 kPa in 400 L of water per hectare. In this paper data on the efficacy of mineral-oil sprays with other insecticides than those of the synthetic pyrethroids will be presented. MATERIAL AND METHODS Plant Material Virus-tested lily bulbs (9-10 cm circumference; 100/plot) of cv. Vivaldi (Asiatic hybrid) were used. ‘Vivaldi’ flowered in the 1st week of July and showed senescence in late September. Plots in three randomised blocks were planted in late March. Each plot had two pairs of five rows (18 cm between rows) of virus-tested lilies separated by one row of LSV+LMoV-infected bulbs of cv. Enchantment (Asiatic hybrid). The trial beds (1 m wide) with the plots were flanked by paths (50 cm) and intermediate beds (1 m) sown with grass. Dahlias were planted (1 m) between the plots. Proc. 8th Int. Symp. on Flowerbulbs Eds. G. Littlejohn et al. Acta Hort. 570, ISHS 2002 278 Chemicals The mineral-oil brand used was Luxan oil H (93 % oil; Asjes and BlomBarnhoorn, 2000) furtherly referred to as ‘Luxan’. The synthetic pyrethroid insecticide was deltamethrin in ‘Decis’ (2.5 % a.i.; 0.0025 % (w/v); 10 g/ha = 400 ml commercial product). The organophosphorus insecticide was acephate in ‘Ypsilon’ (80 % a.i.; 1 kg/ha of commercial product). The pyridine azomethine insecticide was pymetrozine in ‘Plenum’ (25WP; 100 g a.i./ha; 0.4 kg/ha of commercial product). In 1998 ‘Plenum’ was added to the sprays of ‘Decis’ at July 7 and 21, and August 4 and 18 (treatment 4 in Table 2). In 1998 ‘Decis’ was replaced by ‘Plenum’ (treatment 9 in Table 2) at the same dates. In 1999 the same was done at June 29 and July 13 and 27 in the ‘3x-treatments’ (treatment 5 and 8 in Table 2) and additionally at August 10 in the ‘4x-treatments’ (treatment 9 in Table 2). Brand names will be used in Tables 1 and 2. ‘Ypsilon’ was kindly supplied by Luxan BV at Elst and ‘Plenum’ by Novartis Agro Benelux BV at Roosendaal. Application of Sprays Emulsions were prepared shortly before use and then applied with a knapsack sprayer (Birchmeijer Helico nozzles 1.2) at a pressure of 400 kPa and at a volume equivalent to 400 L/ha. Spraying was carried out in calm weather to minimise spray drift. The equivalent dosages were 3.1 (0.78 %) and 6.25 L/ha (1.56 %) for Luxan oil H, 400 mL/ha of Decis, 1 Kg and 1⁄2 Kg/ha of Ypsilon and 0.4 Kg/ha of Plenum. In the Tables half volumes and weights will only be mentioned separately. Sprays were applied weekly in May-July and fortnightly in August-September. Assessment of Bulb Weight and Virus Infection The bulbs harvested in October were stored at 0°C till January-February. They were then washed and dried overnight. The mean weight ratios were calculated as weight of n-treated bulbs: weight of n-control bulbs x 100. The presence of LSV and LMoV in each bulb of Vivaldi was assessed by DSA-ELISA (van Schadewijk, 1986). The rates of infection were calculated for separately infected plants for LSV, LMoV or both. The percentage of spread reduction was calculated by 100 – (rate of virus infection of treated plots : rate of virus infection in untreated plots x 100). RESULTS Table 1 shows weight ratios, percentages of virus infection and reduction of virus spread by mineral oil and the insecticides deltamethrin and acephate. In 1997 and 1998 weight ratios were not significantly different from the data obtained in the untreated plots, except treatment 4 in 1998. In 1999 the infestation by Botrytis troubled weight ratios too strongly to indicate a possible yield-reductive effect of mineral-oil/insecticide sprays. The control by acephate in Ypsilon was less effective than by deltamethrin in Decis, viz., 20-33 % and 41-51 % reduction of virus spread, respectively. The level of efficacy of sprays of Luxan oil H plus deltamethrin was similar to that obtained by Luxan oil H plus acephate. Sprays of Luxan oil H (3.1 l/ha) plus acephate (1/2 kg/ha) were least effective (treatment 9). Table 2 shows data on weight ratios, percentages of virus infection and reduction of virus spread by mineral oil and the insecticides deltamethrin and pymetrozine. The control by single sprays of Plenum was about half or less effective than that obtained by Decis. The control by mixed sprays of Luxan oil H plus Decis or Plenum was similarly effective. DISCUSSION Current-season infection by LSV and LMoV of lily cv. Vivaldi did not evidently affect bulb yields while primary infection of both viruses is traceable from July onwards (Asjes et al., 2000). The potential reduction of bulb yields may also be due to spray treatments, for instance by mineral oils (Asjes, 1984). The potential infestation by

The Asiatic hybrid lily (Lilium L.) 'Connecticut King' harbours several beneficial traits such as partial resistance to Fusarium and complete resistance to tulip breaking virus (TBV). The variation in resistance to Fusarium was determined in four different greenhouse tests in four different years on the same 100 descendants of a backcross population. There was considerable variation in Fusarium resistance levels over the years. TBV resistance was determined and segregated, as expected, like a monogenic trait (1:1). In order to identify quantitative trait loci (QTLs) for Fusarium resistance and also to identify linked markers to the TBV resistance a QTL mapping approach was carried out in this backcross population. Segregation of 399 polymorphic AFLPTM (amplified fragment length polymorphism) fragments was determined. Two different enzyme combinations were used, PstI/MseI and EcoRI/MseI, in the latter one extra selective nucleotide was added to the MseI primer. A paternal ('Connecticut King') linkage map was calculated. This map consisted of 24 linkage groups (Lilium 2n=2x=24) with 251 markers covering 1367 cM. About 25␘f the markers remained unlinked. Four QTLs for Fusarium resistance mapped to linkage groups 1, 5, 13 and 16 respectively. The resistance gene for TBV was placed on linkage group

The nucleotide sequence of the 3′-terminal region of a potyvirus from lily in Miyagi, Japan, exhibited 96.5% homology in the CP gene and 98.9% homology in deduced amino acid sequence to an isolate of Lily mottle virus (LMoV) from the Netherlands. Dilution end-points were compared for the detection of LMoV in lily by an indirect enzyme-linked immunosorbent assay; an assay with immunocapture, reverse transcription (RT) and the polymerase chain reaction (PCR); and an assay with RT and PCR using total RNA extracted from infected plant material. The RT-PCR assay with total RNA was the most sensitive assay which, with primers based on the nucleotide sequence information, was able to distinguish between infection by LMoV and Tulip breaking virus, another potyvirus that can infect lily.

De verspreiding van tulpenmozaïekvirus (TBV) in de tulpenteelt gebeurt door bladluizen en is voornamelijk tegen te gaan door selectie en het bestrijden van de bladluizen soor gewasbespuiting met pyrethroïden. Met de huidige middelen is daarmee een reductie van 50-70% haalbaar. Bij probleempartijen wordt in tulp ook minerale olie ingezet.

Tulip breaking virus (TBV) causes severe economic losses in flower bulbs in the Netherlands. To prevent further spread by aphids, infected plants must be removed from the field as soon as possible. Until now screening is done by visual inspection in the field. As the availability of human experts is limited there is an urgent need for a rapid, automated and objective method of screening. Based on laboratory experiments, we developed a vision method for use in the open field. From 2009 - 2012 field trials were carried out and the techniques were tested and improved. First field trails were on single plant density where plants does not overlap in the images. In 2012 the experiment conducted a tulip field planted at production density of 100 and 125 plants per square meter, resulting in images with overlapping plants. The final score of our system in this production density experiment approached the scores obtained by the experienced crop experts.

Anthers of the Liliumב Enchantment ’ , excised at the uninucleate microspore stage, were cultured on MS media containing 6% sucrose with auxin and cytokinin. When anthers were cultured on the medium with 2 mg l−1 picloram and zeatin, 12–86% of them formed nodular calli. Anthers excised from greenhouse- and field-grown plants showed different responses: anthers of greenhouse-grown plants had a significantly higher capability to form callus and regenerate bulblets than those of field-grown plants. In anthers from greenhouse-grown plants, bulblet formation was dependent on the time at which anthers were excised from donor plants: anthers collected from early forced mother-plants had a higher capability of forming bulblet than others. All regenerated plantlets were diploid, which was substantiated by histological observation showing that the anther-derived calli originated from anther wall tissues. Virus tests by ELISA were made for 49 plantlets selected randomly at transplanting: 20 plantlets (41%) were virus-free, and the rest showed positive reactions for lily symptomless virus, cucumber mosaic virus and/or tulip breaking virus.

The sequence of the 3′-terminal 2074 nucleotides (nts), excluding the 3′-poly (A) tail, of RNA of a potyvirus isolated from lily (Lilium Asiatic hybrid cv. Enchantment) in Japan, currently tentatively designated as Tulip breaking virus-li (TBV-li), was determined. The sequence started within a single open reading frame (ORF) that encoded the carboxyl terminus of the large nuclear inclusion protein (NIb) and the complete 275-amino-acid coat protein (CP), followed by a 3′-untranslated region (3′-UTR) of 204 nts. The CP of TBV-li shared 91% amino acid (aa) sequence identity with that of TBV lily strain Dutch isolate (TBV-lily). The nt sequences of their 3′-UTR were 94% identical. However both viruses shared only 60–65% sequence identities with TBV tulip strain Niigata isolate in the corresponding regions. The results suggest that TBV-li is closely related to TBV-lily, and that these two TBV lily strains should be classified into a species different from TBV tulip strains. We therefore support a proposal to rename TBV-lily Lily mottle virus (LMoV), and suggest that TBV-li is another strain of LMoV (LMoV-J).

This article focuses on the oldest recorded plant virus disease, tulip breaking, and reflects the authors’ interests in molecular virology, the history of virology, and the broad influence of viruses upon societies and their culture. The potyvirus Tulip breaking virus (TBV) induces in the petals of its host tulips beautiful variegated color patterns that break the solid color of the uninfected tulips, hence the name “tulip breaking.” The human passions of possession that these “broken tulips” induced in seventeenth century Holland generated an economic and social disorder with lasting cultural ramifications referred to as “tulipomania.” Although the lure of the broken tulip persists in the twenty-first century, the molecular mechanisms governing the virus-induced color breaking in tulips remain little understood. Here, we review aspects of the historical impact of tulipomania, the biology of TBV, the pathways and regulation of plant pigment formation, and the potential mechanisms underlying virus-induced color breaking. The reader is cautioned that tulipomania, like tulip breaking, is still contagious.

1. The viruses, LSV, TBV-L, and CMV were made in the scale-segments of Lilium X 'Enchantment' by both a dot immunobinding assay (DIBA) and indirect enzyme-linked immunosorbent assay (ELISA). The sensitivity of DIBA to detect viruses is about 10 to 100 times lower than that of ELISA, but it is an easy, reliable method to detect these viruses in routine surveys. 2. Absorbance values of LSV in leaves which were collected from the lower, middle, and upper parts of stem, perianths, and bracts were determined at 405 nm by ELISA. The most reliable value was obtained from the basal parts of leaves, located on the upper part of the stem. Based on this result, the absorbance of leaves collected at various times during the growth period was determined. The LSV level in the leaves increased gradually toward anthesis. Whereas the leaves excised from the upper part of the stem had a higher concentration of LSV than had those of the lower part in L. X 'Enchantment' and L. X 'Star Gazer', the reverse trend was found in L. formosanum. 3. Individual plants (550) of 18 Lilium spp. growing in the field were examined for three viruses by DIBA. Leaves of test plants were collected from the upper part of stems at anthesis and their basal parts were analyzed for three viruses. One or more viruses were detected in 73% of the 550 plants investigated. The frequency of LSV, TBV-L, and CMV occurring alone in the population was 52, 2, and 1%, respectively ; the remaining 18% was infected with two or three viruses. More than 80% of the plants in 10 Lilium spp. were infected with one or more viruses. 4. Lilium spp. infected with LSV were nearly symptomless. Plants of L. rubellum with the leaves which develop green stripe mosaic were infected with LSV and TBV-L. Stem elongation of plants of 'Casablanca', 'Hinomoto', 'Enchantment', and 'Star Gazer' multiple-infected with LSV and TBV-L or CMV was retarded compared with stem elongation of the same cultivars infected with LSV alone.

Tulpenmoza¿ekvirus (TBV) in tulp veroorzaakt veel schade in de teelt. Om virusverspreiding door bladluizen te voorkomen moet de teler in een korte periode deze bronnen van infectie verwijderen. Een tijdrovende bezigheid, die ook specifieke deskundigheid vergt. Verder bespaart dit verwijderen ook het gebruik van gewasbeschermingsmiddelen om de luizen te bestrijden. Echter, experts die dit kunnen zijn schaars. Om het gebrek aan goede ziekzoekers te compenseren, werkt Wageningen UR samen met een groep tulpentelers aan een oplossing. Gerobotiseerd ziekzoeken moet in een vroeg stadium symptomen van ziekten (TBV) in het gewas (in dit geval tulp) kunnen herkennen. Het streven is dat dit minimaal even goed gebeurt als door de experts.

Tulpenmoza¿ekvirus (TBV) in tulp veroorzaakt veel schade in de teelt. Om virusverspreiding door bladluizen te voorkomen moet de teler in een korte periode deze bronnen van infectie verwijderen. Een tijdrovende bezigheid, die ook specifieke deskundigheid vergt. Verder bespaart dit verwijderen ook het gebruik van gewasbeschermingsmiddelen om de luizen te bestrijden. Echter, experts die dit kunnen zijn schaars. Om het gebrek aan goede ziekzoekers te compenseren, werkt Wageningen UR samen met een groep tulpentelers aan een oplossing. Gerobotiseerd ziekzoeken moet in een vroeg stadium symptomen van ziekten (TBV) in het gewas (in dit geval tulp) kunnen herkennen. Het streven is dat dit minimaal even goed gebeurt als door de experts.

Tulip breaking virus (TBV) threatens bulb and flower production of tulips seriously. Breeding for resistance as a tool to prevent this disease resulted already in a screening test at clonal level and different levels of resistance were detected. Clonal tests are performed under greenhouse conditions after inoculation by viruliferous aphids. By using several bulbs of a genotype an accurate level of resistance can be determined. All cultivars of T. gesneriana tested were susceptible, but partial and even absolute resistance was found in T. fosteriana cultivars. To introduce resistance from T. fosteriana in the T. gesneriana assortment interspecific crosses were made. To select TBV resistant hybrids efficiently, a screening test, applicable at individual seedlings, is described. To test individual seedling selection, an incomplete diallel was made between the susceptible T. gesneriana cultivars ‘Christmas Marvel’, ‘Kees Nelis’ and ‘Lustige Witwe’, the partial resistant T. fosteriana cultivars ‘Juan’ and ‘Madame Lefeber’ and the absolute resistant T. fosteriana cultivars ‘Cantata’ and ‘Princeps’. Individual seedlings were inoculated using viruliferous aphids in the first, third or fifth year after sowing. Six weeks after inoculation, leaves were tested for TBV by ELISA. All seedlings were grown till maturity and the occurrence of breaking symptoms in the flower was monitored. Individual resistant seedlings could be selected. However, susceptible plants can occur (escapes) between the selected plants and also resistant plants can be discarded (missings). The efficiency of this seedling test will be discussed.

De teelt van tulpen kampt met aantasting door verschillende virussen, met name door het mozaïekvirus (Tulip Breaking Virus of TBV). Virusaantasting uit zich in de vorm van vaak subtiele strepen, vlekken in het blad en kleurafwijkingen in de bloemen. De aanwezigheid van het virus in de tulpenbol verlaagt de opbrengst en de kwaliteit en is een belemmering voor de export. Bij een hoge besmetting worden hele partijen afgekeurd. om economische, arbeid technische én milieukundige redenen gewenst virusaantasting zoveel mogelijk te voorkomen. Een consortium van kwekers, WUR-PPO, Agro-Syntens met steun van de sector (KAVB) heeft als doelstelling om het percentage viruszieke tulpen in de gehele keten terug te dringen door middel van het ontwikkelen en testen van een autonoom werkend prototype apparaat voor detectie en verwijdering van virus besmette (tulpen) planten in het open veld.

De teelt van tulpen kampt met aantasting door verschillende virussen, met name door het mozaïekvirus (Tulip Breaking Virus of TBV). Virusaantasting uit zich in de vorm van vaak subtiele strepen, vlekken in het blad en kleurafwijkingen in de bloemen. De aanwezigheid van het virus in de tulpenbol verlaagt de opbrengst en de kwaliteit en is een belemmering voor de export. Bij een hoge besmetting worden hele partijen afgekeurd. om economische, arbeid technische én milieukundige redenen gewenst virusaantasting zoveel mogelijk te voorkomen. Een consortium van kwekers, WUR-PPO, Agro-Syntens met steun van de sector (KAVB) heeft als doelstelling om het percentage viruszieke tulpen in de gehele keten terug te dringen door middel van het ontwikkelen en testen van een autonoom werkend prototype apparaat voor detectie en verwijdering van virus besmette (tulpen) planten in het open veld.

Tulip breaking virus (TBV) causes severe economic losses for the Netherlands. Infected plants must be removed from the field as soon as possible to prevent further spread by aphids. Until now screening is done by visual inspection in the field. As the availability of human experts is limited there is an urgent need for a rapid, automated and objective method of screening. Based on laboratory experiments in 2008, we developed a vision method for use in the open field. From 2009 – 2011 field trials were carried out and the techniques were tested and improved. The final score of our system in the last experiment (2011) approached the scores obtained by the human crop experts.

Vorig jaar herkenden de camera’s en software van de ziekzoekkar TBV-symptomen in de tulpencultivar ‘Yokohama’ bijna net zo goed als de ervaren ziekzoeker. Echter, de tulpen waren op lage dichtheid geplant. Dit jaar was de doelstelling of de ziekzoekkar volvelds virusziekte ‘Yokohama’ kon herkennen en dan minstens net zo goed als de deskundige ziekzoekers. Door aanpassingen aan belichting en de inbouw van speciale camera’s scoorde de ziekzoekkar beter dan de ervaren ziekzoekers: een doorbraak!

Tulip breaking virus (TBV) causes severe economic losses in flower bulbs in the Netherlands. To prevent further spread by aphids, the vector of the disease, infected plants must be removed from the field as soon as possible. Until now screening has been carried out by visual inspection in the field. As the availability of human experts is limited there is an urgent need for a rapid, automated and objective method of screening. Based on laboratory experiments, a vision method for use in open fields has been developed. In the period 2009e2012 field trials were carried out and the techniques were tested and improved. During the final evaluation of our system, in the last experiment (2012), the system approached the scores obtained by the experienced crop experts.

In 2008 is het project Ziekzoeken in tulp gestart na een succesvol laboratoriumexperiment. In 2009 bleek de overgang van laboratorium naar een handmatig bediend prototype in het veld een te grote stap. In 2010 zijn de camera's, belichting en uitvoering van de ziekzoekrobot aangepast. Uit de resultaten in 2010 bleek dat een verdere verbetering van de ziekzoekrobot noodzakelijk was. De veranderde cameraopstelling, scherptediepte van de opnames en belichting zijn in 2011 opnieuw getest. Nu blijkt dat de ziekzoekrobot bijna net zo goed scoort als zeer ervaren ziekzoekers.

Virus transmission by aphids causes millions of Euro’s damage in the flower bulb sector annually. Mineral oils and pyrethroids are applied weekly during the growth season to decrease the virus transmission by aphids in flower bulbs. Currently, little is known about the dynamics of aphid populations during the growth season of tulips and the accompanying risk of the non-persistent virus transmission of for example Tulip breaking virus (TBV). Therefore, population dynamics of aphids in tulip fields was monitored during three growing seasons. Simultaneously, the period of TBV transmission by aphids was investigated experimentally by exposing small tulip plots during specified intervals to naturally occurring aphids. Finally, timing of virus spread was correlated with aphid population dynamics and weather conditions. In 2007, TBV transmission started in April while the first aphids were found in May. Apparently, the first aphids of the season already contributed in great extend to the virus transmission and virus transmission was observed very locally in the tulip plots. A second, distinct TBV isolate was identified which resulted in the design of an improved TBV detection assay. The main objective of this research project is to enhance the knowledge about the risk of non-persistent virus transmission in relation to aphid population dynamics. This knowledge should enable us to formulate crop protection guidelines that are better fine-tuned with the risk of virus transmission and thereby decreasing the environmental damage caused by chemical crop protection.

Five viruses causing colour-breaking of tulip flowers were isolated from tulips and lilies. Tulip-breaking virus (TBV), tulip top-breaking virus (TTBV), tulip band-breaking virus, Rembrandt tulip-breaking virus and lily mottle virus were all characterized as potyviruses by serology and potyvirus-specific PCR. Sequence analysis of amplified DNA fragments spanning a conserved area of the coat protein cistron of potyviruses was performed in order to classify the isolates as distinct viruses or strains. It appears that all tulip-breaking viruses are distinct viruses and TTBV was found to be strain-related to turnip mosaic virus.

De verspreiding van tulpenmozaïekvirus (TBV) in de tulpenteelt gebeurt door bladluizen en is voornamelijk tegen te gaan door selectie en het bestrijden van de bladluizen soor gewasbespuiting met pyrethroïden. Met de huidige middelen is daarmee een reductie van 50-70% haalbaar. Bij probleempartijen wordt in tulp ook minerale olie ingezet.

In 2009 heeft de ziekzoekrobot voor het eerst op PPO onderzoek gedaan naar viruszieke tulpen. Ervaren ziekzoekers gingen eveneens aan de slag. De resultaten van de cultivar 'Barcelona' zijn geanalyseerd en hebben geleid tot enige aanpasssingen in het onderzoek dat in 2010 wordt voortgezet.

In tulpen veroozaakt het tulpenmozaïekvirus van alle virussen de meeste schade. PPO Bloembollen onderzocht wanneer de virusverspreiding plaatsvindt, welke bladluizen een rol spelen en hoe het risico op virusverspreiding het beste vooraf in te schatten is. Dit heeft geleid tot verrassende resultaten, die vragen om een aangepaste bestrijding. Het is duidelijk geworden dat gewasbescherming tegen virusoverdracht al moet starten zodra de temperatuur rond de 12-13° komt

The inhibitory effect on the spread of viruses in lilies viz., tulip breaking virus (TBV; nonpersistently aphid-borne, potyvirus,) lily symptomless virus (LSV; non-persistently alphidborne, carlavirus), and lily virus X (LVX; potexvirus of unknown etiology), was studied of brands of mineral oil (Luxan oil H and Duphar-7E oil) and synthetic pyrethroid insecticides (l-cyhalothrin and deltamethrin), and a nematicide (aldicarb) in crops in which virus-infected plants were present as virus sources. The spread of TBV and LSV were controlled by sprays of mineral oil and insecticide, while that of LSV was also limited by the soil-applied nematicide. The spread of LVX was reduced by the insecticides and, not effectively by the mineral-oil spraying, by which data the mode of transmission may be presumed to be by an insect in the persistent or semi-persistent manner. Mixtures of mineral oil and pyrethroid were more effective in the reduction of spread of TBV and LSV than either components tested alone. The mineral oil was the most effective component in the mixtures in which pyrethroid added a slight extra effect. The addition of pyrethroid did not mask either the lower efficacy of the oil brand Duphar-7E oil, or the diminished inhibitory effect of low dosages of oil. The normal rate of mineral oil gave similar control to that of a mixture of mineral oil at half rate plus the pyrethroid at full dosage. Low rates of oil, or even synthetic pyrethroids alone may be used on cultivars which suffer of the loss of bulb weight by the use of normal or decreased rates of oil. Weekly sprays were more effective than fortnightly sprays. The rate of control by the weekly sprays ranged between 90 and 95% for Luxan oil H at half dosage plus the full rate of pyrethroid. Weekly sprayed synthetic pyrethroids alone onto the virus sources and the plants to be infected gave 60–70% control. The weight ratios tended to be slightly reduced if the half dosage of the efficient Luxan oil H was used. Factors which affect the control of the air-borne field spread of viruses by mineral oils and synthetic pyrethroid insecticides in lilies are discussed.

Sunflower mosaic is caused by a putative member of the family Potyviridae. Sunflower mosaic virus (SuMV) was characterized in terms of host range, physical and biological characteristics, and partial nucleotide and amino acid sequence. Cells infected with SuMV had cytoplasmic inclusion bodies typical of potyviruses. Of 74 genera tested, only species in Helianthus, Sanvitalia, and Zinnia, all Asteraceae, were systemic hosts. Commercial sunflower hybrids from the United States, Europe, and South Africa were all equally susceptible. The mean length of purified particles is approximately 723 nm. The virus was transmitted by Myzus persicae and Capitphorus elaegni, and also was seedborne in at least one sunflower cultivar. Indirect enzyme-linked immunosorbent assay tests with a broad-spectrum potyvirus monoclonal antibody were strongly positive. SuMV-specific polyclonal antisera recognized SuMV and, to a lesser extent, Tobacco etch virus (TEV). When tested against a panel of 31 potyvirus-differentiating monoclonal antibodies, SuMV was distinct from any potyvirus previously tested. SuMV shared four epitopes with TEV, but had a reaction profile more similar to Tulip breaking virus (TBV). SuMV did not possess epitopes unique only to TBV. The predicted coat protein had a molecular weight of 30.5 kDa. The 3′ end of the virus genome was cloned and sequenced. Phylogenetic analysis of the coat protein amino acid sequence revealed that SuMV is a distinct species within the family Potyviridae, most closely related to TEV.

In tulpen veroorzaakt het tulpenmozaïekvirus (Engels: Tulip Breaking Virus, TBV) van alle virussen de meeste schade. Er is directe schade zoals opbrengstverlies en kwaliteitsverlies veroorzaakt door virussymptomen. Daarnaast is er indirecte schade veroorzaakt door de beheersingsmaatregelen en verplichte keuringsmaatregelen. TBV behoort tot de familie van de potyvirussen, virussen die vooral door bladluizen of andere insecten worden overgebracht. Vooral in de gele (en witte) tulpencultivars is het virus steeds moeilijker onder controle te krijgen. Percentages TBV van 6% en hoger, waarbij virusbeheersing vrijwel onmogelijk is geworden, zijn geen uitzondering meer. Dit heeft onder anderen te maken met de schaalvergroting van bedrijven, waardoor er minder tijd en expertise beschikbaar is voor het ziekzoeken, de slechte of in de tijd zeer beperkte zichtbaarheid van symptomen (typerend voor gele en witte cultivars) en mogelijk de grotere vatbaarheid van deze cultivars voor TBV. Het verwijderen van virus(bron)planten gedurende de teelt door middel van ziekzoeken levert wel het hoogste rendement op in de virusbestrijding: het viruspercentage kan daardoor worden verlaagd en er is minder kans op virusverspreiding! Dit twee jarig onderzoek richt zicht op het beperken van TBV-verspreiding gedurende de teelt van de vooral gele tulpencultivars. Om dit te bereiken zijn de experimenten gericht op het bepalen van de mate van virusverspreiding onder invloed van diverse teelthandelingen en –omstandigheden.

In tulpen veroorzaakt het tulpenmozaïekvirus van alle virussen de meeste schade. PPO Bloembollen heeft twee jaar onderzocht wanneer en hoe virusverspreiding plaatsvindt en hoe dit te beperken is