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Tulip bulb and flower production greatly contribute to the economy in The Netherlands. However, severe loss in this industry is caused by different viruses. In recent years, diagnostic tools were developed for most of these viruses with Tulip severe mosaic virus (TSMV) as one of the few exceptions. To further characterize TSMV, the virus was amplified by reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers targeted to conserved sequences of the Heat shock protein 70 gene of viruses from the family Closteroviridae. Phylogenetic analysis of the 0.5 kb HSP70 gene fragments showed that TSMV is closely related to Plum bark necrosis stem pitting-associated virus (PBNSPaV), Apricot stem pitting-associated virus (ASPaV) and Pineapple mealybug wilt-associated virus 1 (PMWaV-1), all three members of the genus Ampellovirus. In addition, a set of primers amplifying a TSMV-specific fragment was designed that enables fast and specific detection of the virus. Additionally to the molecular experiments, two possible modes of virus transmission were tested, viz. by aphids and by soil-borne organisms. However, no transmission was recorded by both methods.

Lily symptomless virus (LSV) and lily mottle virus (LMoV) are detected reliably with ELISA in bulbs of Asiatic hybrids stored at 0-2°C for at least 2-3 weeks after lifting prior to testing. If this protocol was applied to Lilium longiflorum-cultivars and Oriental hybrids, however, it generally failed in the detection of LMoV. This phenomenon was virus dependent since LSV, cucumber mosaic virus and lily virus X were easily detected. The detection of LMoV in L. longiflorum-cvltivais greatly improved after storage of scales at 20°C for 2-3 weeks under white fluorescent light (12-16 h/day, 13-16 W.m-2). This protocol was also applicable to LSV in L. longiflorum, but not for LMoV in Oriental hybrids.

In dahlia visual selection on tomato spotted wilt virus (TSWV) is only possible to a limited extent. The unequal distribution of the virus in various plant parts during the growing and storage period caused problems in the development of a reliable test protocol. A serological test on tubers appeared to be the best: the tubers have to be stored for at least 1 month at 9°C and samples have to consist of at least three roots per tuber. 40-90% of the cuttings from TSWV-infected dahlia tubers appeared to be infected. Symptoms of TSWV in both gladiolus and iris cannot be distinguished from those caused by Impatiens necrotic spot virus (INSV). In iris a third (putative) tospovirus was detected, which is called iris yellow spot virus. In progeny-plants from infected iris and gladiolus no tospoviruses were detected. In Hippeastrum, Amaryllis and Nerine TSWV was detected in symptom-bearing leaves and also in the tunics connected with these leaves, but less frequent in other parts of the bulbs. This might explain the curing of the main bulbs after some years and the limited transmission of TSWV to the daughter bulbs.

Potyviruses (genus Potyvirus, family Potyviridae) are transmitted by aphids in a non-persistent manner and cause significant losses in many crops including bulbous ornamentals. Host range, symptoms, physical and biochemical properties of many potyviruses in bulbous ornamentals are reported, but, especially for viruses infecting ornamentals of minor economical importance, sequence data are still lacking. We used molecular techniques for the identification, characterization and detection of these viruses. Leaf material of several ornamental crops showing virus-like symptoms were tested in indirect ELISA, using monoclonal antibodies specific for potyviruses. Generic potyvirus primers were used in an RT-PCR to amplify the 3' terminal region of these viruses. The fragments encode the viral coat protein (CP) gene and comprise the 3'-untranslated region (3'-UTR). Nucleotide sequences of the obtained fragments were determined and compared with potyvirus sequences present in the NCBI database using the BLAST algorithm. We have characterized some potyviruses previously accepted by the International Committee on Taxonomy of Viruses (ICTV), including Freesia mosaic virus (FreMV), Gloriosa stripe mosaic virus (GSMV), Hippeastrum mosaic virus (HiMV), Hyacinth mosaic virus (HyaMV), Iris mild mosaic virus (IMMV) and Nerine yellow stripe virus (NeYSV). Additionally, the identities of other potyviruses infecting ornamentals such as Anemone, Galtonia, Muscari, Ornithogalum, Allium, Stenomesson and Veltheimia have been determined. These viruses, however, have not yet been reported by the ICTV. The virus-specific sequence information generated in this research project can subsequently be used to develop PCR-based detection methods.

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.

Summary The influence of wounding and high-temperature treatment on the detection of iris severe mosaic virus (ISMV) in secondarily ISMV-infected iris bulbs was studied. Wounding of the bulbs just after lifting, followed by storage for 3 wk at 17°C or 20°C, increased the detectability of ISMV to 100% reliability. High-temperature treatment and consecutive storage at 17°C induced a similar improvement of detection. It is concluded that a certain degree of stress, such as wounding or high-temperature treatment, ultimately leads to an increase in viral antigens and thus to improvement of detection. It is hypothesised that the virus titre increases by the altered metabolism during the repair reactions as a response to stress applied to the bulbs.

cDNA clones complementary to the 3'-terminal regions of the genomic RNAs of the carlavirus lily symptomless virus and the potexvirus lily virus X (LVX) have been sequenced. The carlavirus RNA sequence contains five open reading frames (ORFs) coding for proteins of Mr 25,374, 11,631, 6960, 32,041 (coat protein) and 16,121, which are all very similar in size, amino acid sequence and relative position in the genome to proteins encoded by two different potato carlaviruses. The first four of these proteins also show considerable amino acid sequence similarity to proteins encoded by RNA of potexviruses, and the relative position of the ORFs on the carlavirus genome strongly resembles that in the potexvirus genomes. The LVX cDNA clone contains three ORFs encoding proteins of Mr 23,574, 11,767 and 21,569 (coat protein). A small ORF immediately 5' of the coat protein ORF, which has been found in other potexvirus genomes, is not present in the LVX genome. Thus, the data confirm the close taxonomic relationship between carlaviruses and potexviruses and reveal some differences in genome organization among the potexviruses.

The coat protein (CP) genes and flanking regions of three alstroemeria-infecting cucumber mosaic virus isolates (CMV-ALS), denoted ALS-LBO, ALS-IPO, and ALS-NAK, were cloned and their nucleotide sequence determined and compared at both nucleic acid and deduced protein level with the published sequences of CMV RNA 3. The sequences of these isolates showed more than 95% nucleotide and peptide sequence homology to each other and to other members of subgroup II CMV. Strikingly, an additional sequence of 218 nt was found in the central region of the 3’-non-translated region (NTR) of RNA 3 of two out of three isolates. The additional sequence appeared to have arisen from RNAs 1 or 2. A subgroup-specific DIG-labeled probe has been developed from this additional sequence and applied to detect subgroup II CMV strains in dot blot hybridization and to differentiate the Alstroemeria isolates containing additional sequences in Northern blot hybridization.

Mixed infections of potyviruses and other viruses occur very frequently in Narcissus. Isolation and characterization of individual potyviruses from Narcissus has partially failed sofar, because other host plants have not been found. In the Netherlands, three different potyviruses from Narcissus were distinguished on the basis of symptomology, viz. narcissus yellow stripe virus, narcissus silver streak virus and narcissus late season yellows virus. We have applied general PCR on various Narcissus samples to identify these viruses at the molecular level and to establish their relationships by sequence analysis of parts of their coat protein cistrons. We conclude that at least three different potyviruses occur in Dutch stocks of Narcissus and that narcissus silver streak virus is identical to narcissus late season yellows virus. Of the other two potyviruses, one occurred in N. tazetta and is probably identical to the narcissus degeneration virus described in England. A third potyvirus was detected together with NLSYV in a sample with symptoms of 'yellow stripe'.

The ISMV isolates studied so far, have been found to be indistinguishable both by serological and hybridization assays. Therefore, the different symptoms induced by these isolates may be based on only minor molecular changes.

The determination of relationships among and the identification of potyviruses with polyclonal antibodies does not always lead to a proper conclusion. The potyvirus specific monoclonal PTY 1 does not detect all potyviruses tested.

Local areas of conserved amino acid sequence in the replicase and coat proteins of potyviruses were used to select nucleotide sequences for use in the construction of sets of degenerate oligonucleotide primers for amplification of DNA fragments on potyvirus-specific templates in a combined assay of reverse transcription and the polymerase chain reaction (RT-PCR). Sequences selected for the construction of degenerate primers included the coat protein gene sequence of tulip breaking virus from lily, which is reported in this paper. It is shown that the degenerate primers support potyvirus-specific amplification, but do not support amplification on carlavirus and potexvirus templates. A panel consisting of definite and prospective members of the potyvirus group occurring in bulbous crops was subjected to the degenerate primer RT-PCR assay; amplified fragments were used in cross-hybridization experiments and restriction fragment length polymorphism analysis to detect relationships among these potyviruses. A partially characterized virus isolated from Gloriosa rothschildiana was positively identified as a potyvirus by specific amplification and subsequent sequence analysis of an amplified DNA fragment.

After comparison of various homogenization buffers and clarification methods, tulip breaking virus (TBV) was purified on a large scale by homogenization in 0.1 M tris buffer (pH 9.0), clarification with Triton X-100, and differential centrifugation. The TBV suspensions were absorbed with an anti-host protein antiserum. The purity of the product was verified by mechanical inoculation of test plants, serology, electron microscopy, spectrophotometry, and analytical ultracentrifugation.

In freesia cv. Aurora grown in the field for cutflower production, a disease occurred with symptoms of leaf-yellowing in combination with corm necrosis (LYCN). It is shown that this disease is caused by bean yellow mosaic virus (BYMV). No differences in symptoms of LYCN were observed between the freesia cultivars Aurora, Imperial and Rose Marie. Most BYMV isolates gave rise to LYCN; the isolates from crocus andIxia sp. did not. LYCN was stimulated by a high BYMV concentration in the inoculum, a temperature above 20°C, inoculation soon after emergence of the freesias, and by the absence of freesia mosaic virus. Freesias with mosaic symptoms and infected with a cross-protecting BYMV strain, did not show symptoms of leaf-yellowing and/or corm necrosis after inoculation with BYMV-Cm. The presence of the unknown agent causing leaf necrosis in freesias did not have an influence on symptom development after infection with BYMV.

Detection of iris severe mosaic virus (ISMV) in ISMV-infected iris bulbs during storage, by either ELISA or electron microscopy has been problematic. We have applied different storage-temperature treatments and a cutting method as possible procedures to enhance ISMV detection in stored iris bulbs cv. Professor Blaauw. While at lifting ISMV could not be detected in the bulbs, gradually the virus became detectable in time when bulbs were stored at about 17°C. Better results were obtained when the cutting method was employed. Using ELISA, a 100 ␜core was obtained for infected bulbs that were cut 1 month prior to testing. The virus could be detected only in tissue adjacent to the cut surface. These results offer good prospects for the development of a reliable detection method for ISMV in iris bulbs.

Lily symptomless virus (LSV) was purified by clarification with chloroform, precipitation with polyethylene glycol and NaCl, and differential centrifugation. The influence of the source material and some buffers on virus yields were determined. Antisera were prepared against intact and pyrrolidine degraded LSV. It was concluded that intact and degraded LSV have very few antigenic determinants in common or none at all. The sensitivities of the micro-precipitin test and the single immunodiffusion drop test were about the same, but lower than that of electron microscopy. In the testing of lilies for LSV the most reliable results in leaves were obtained during the period from two weeks after flowering until close to the end of the growing season, and in leaves growing at a level about one-fourth of the distance from the top of the stem. In contrast to secondary infections, primary infections were detected more successfully in stored bulbs than in leaves taken from plants in the preceding growing season. In the testing of tulips, LSV was detected better in flowers than in leaves. Detection of primary infections was almost impossible. Except for those with a pink flower, experimentally infected tulips remained symptomless. Het symptoomloos lelievirus (LSV) were gezuiverd door klaring met chloroform, precipitatie met polyethyleenglycol en NaCl en differentieel centrifugeren. Het effect van het uitgangsmateriaal en enkele buffers op de virusopbrengst werd nagegaan. Antisera werden bereid tegen intact en met pyrrolidine afgebroken LSV. Geconcludeerd were dat intact en afgebroken LSV geen of slechts enkele antigene determinanten gemeenschappelijk hebben. De gevoeligheid van de microprecipitatietoets en de enkele immuno-diffusiedruppeltoets is ongeveer gelijk. Met het elektronenmicroscoop kunnen echter lagere virusconcentraties worden aangetoond. Het toetsen van lelies op LSV gaf de betrouwbaarste resultaten met bladeren die op ongeveer driekwart hoogte van de stengel groeien en in de periode tussen 2 weken na de bloei en vrijwel het einde van het groeiseizoen worden geplukt. Primaire infecties konden, in tegenstelling tot secundaire infecties, beter worden vastgesteld aan bolmateriaal tijdens de bewaring dan aan bladeren in het voorafgaande groeiseizoen. Bij het toetsen van tulpen werd het LSV met grotere zekerheid vastgesteld in bloemen dan in bladeren. Het vaststellen van primaire infecties was bijna niet mogelijk. Na infectie van tulpen met LSV vertoonden alleen die met een rose bloemkleur symptomen.