Variability of Peronospora Sparsa (syn. P. rubi) in Finland as Measured by Amplified Fragment Length Polymorphism

Downy mildew caused by Peronospora sparsa (syn. P. rubi) is a serious threat to commercial cultivation of arctic bramble (Rubus arcticus subsp. arcticus) in Finland. P. sparsa is distributed throughout the country in cultivated and wild arctic bramble and in cloudberry (R. chamaemorus). A total of 36 isolates of P. sparsa collected from these hosts was analysed for amplified fragment length polymorphism (AFLP). Of the 226 markers scored, 223 were polymorphic and all isolates of P. sparsa had unique AFLP fingerprints, which indicated high levels of genetic variability. An UPGMA clustering analysis of the isolates did not reveal any genetically distinguishable strains. The isolates were grouped neither according to the geographic origin nor the host from which they were isolated. Isolates of P. sparsa obtained from wild arctic bramble and one from cloudberry readily infected the leaves of the cultivated arctic bramble (cultivar Pima). Also, P. sparsa isolated from cultivated arctic bramble infected the leaves of wild arctic bramble. These data suggest that P. sparsa may be disseminated from wild arctic bramble and cloudberry to cultivated arctic bramble in the field.     

Phoma–Didymella complex on hybrid arctic bramble with wilting symptoms

Pycnidia containing conidia characteristic of Phoma spp. and pseudothecia containing ascospores characteristic of Didymella applanata were isolated from edges of expanding stem lesions and dead stems of wilted cultivated hybrid arctic bramble plants ( Rubus arcticus nothossp. stellarcticus ) in Sweden in 1998 and 1999. The fungi were morphologically similar when grown on culture media, but some differences in growth rate were observed. They were also similar to the reference isolates of D. applanata (anamorph Phoma argillacea ). However, they were different from an isolate of Phoma sp. (HPP 38) isolated from cultivated arctic bramble ( R. arcticus ssp. arcticus ) in Finland in 1980, and from reference isolates of Phoma glomerata isolated from other hosts. Multivariate analysis of growth rate data and conidial dimensions measured in vitro indicated that the fungi isolated from hybrid arctic bramble in Sweden were not distinguishable from D. applanata , but were clearly distinct from P. glomerata and P. exigua. Furthermore, they had identical ITS1 and ITS2 sequences, and were placed in a phylogenetic clade very closely related to the clade that contained isolates of D. applanata isolated from raspberry ( Rubus idaeus ). In contrast, isolate HPP 38 from Finland was placed in a clade with P. exigua. These data indicate that the Swedish isolates infecting arctic bramble belong to a strain of D. applanata that differs from the isolate infecting raspberry only by two common nucleotide substitutions in ITS2. Fungi of the Phoma–Didymella complex on wild and cultivated arctic bramble (a total of 291 plants showing symptoms sampled from 37 sites) were detected by a PCR-based assay and found to be common in northern Sweden, but rare, albeit widely distributed, in Finland.     

Quantification of downy mildew (Peronospora sparsa) in Rubus species using real-time PCR

Downy mildew disease caused by Peronospora sparsa, also known as ‘dryberry’ disease, is a serious threat to the cultivation of arctic bramble (Rubus arcticus) and boysenberry (Rubus spp. hybrid). A quantitative and sensitive screening method is necessary for the breeding of downy mildew resistant cultivars and for determining efficient disease control methods. A quantitative real-time PCR method using SYBR^® Green I fluorescent dye was developed for the analysis of P. sparsa in arctic bramble, other Rubus species and roses. Primers were designed to amplify a P. sparsa specific 94-bp fragment from the internal transcribed spacer region (ITS1) and a 140-bp fragment from a conserved region of plant 5.8S ribosomal DNA, which served as an internal control in the samples. Linear amplification from genomic DNA and control plasmids was achieved with both primers, and even 37 fg of P. sparsa conidial DNA was detected. In the samples collected from the field, quantities as low as 0.2 ppm of P. sparsa DNA in plant DNA were detected, thus enabling the diagnosis of weak and latent infections. Arctic bramble cvs Pima, Mespi and Mesma, all showing distinct foliar symptoms, were tested to assess the relative amount of downy mildew DNA present. The symptoms and the amount of P. sparsa DNA detected correlated only in cv. Pima, indicating that visual inspection of symptoms is not a reliable method for assessing the extent of tissue infection. The number of conidiophores of P. sparsa on in vitro inoculated leaves of two arctic bramble cultivars correlated with the results obtained by real-time PCR.     

Association of three RNA molecules with potato mop-top virus

Summary Potato mop-top virus particles, purified from systemically infectedNicotiana benthamiana plants and then disrupted by heating with sodium dodecyl sulphate and 2-mercaptoethanol, contained only a single polypeptide of M_r 19 100 detectable by polyacrylamide gel electrophoresis. Single-stranded RNA preparations from virus particles, when subjected to electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturant, were shown to contain approximately equal proportions of three RNAs of 6.5, 3.2 and 2.5 kb. Double-stranded RNA preparations extracted from systemically infectedN. benthamiana leaves or from locally infectedN. debneyi leaves, was shown by polyacrylamide gel electrophoresis to contain a major species of 3.2 kbp and two minor species of 6.5 and 2.4 kbp.     

First report of raspberry yellows disease caused by raspberry bushy dwarf virus in Japan

Severe yellowing of leaves was observed on red raspberry in Akita Prefecture. When inoculated with sap from symptomatic raspberry leaves, Chenopodium quinoa plants developed chlorotic ringspot and mottling that are typical of raspberry bushy dwarf virus (RBDV) infection. In western blot analysis, an antibody to the coat protein (CP) of RBDV reacted against ca. 30-kDa protein specific to the diseased trees. In RT-PCR testing for RBDV, single DNA fragments were amplified from total RNA samples of the diseased trees. The nucleotide sequences of the DNA fragments covering the entire CP region revealed 87–97?% identities with those of RBDV isolates.     

Een virus inAtropa belladonna

Summary In a bgarden at Baarn, The Netherlands,Atropa belladonna plants, grown from seed, showed symptoms similar to those described bySmith (1946, 1957) for Belladonna mosaic. After inoculation of solanaceous test plants with sap from diseased plants, the following species showed symptoms:Atropa belladonna L.,Capsicum annuum L.,Hyoscyamus niger L.,Nicandra physaloides Gaertn.,Nicotiana glutinosa L.,Nicotiana tabacum L. var. Samsun,Petunia hybrida andPhysalis floridana Rydb. The symptoms suggest that the virus may be identical with that described bySmith. A high virus concentration was found inHyoscyamus niger. Nicandra physaloides, Petunia hybrida, Physalis floridana, in the roots and the pericarp of diseasedAtropa plants, and also inSolanum nigrum L. andDatura stramonium L. The latter two species showed hardly any symptoms. A. low virus concentration was found inCapsicum annuum, though this plant showed severe symptoms.The dilution end point of the virus was between 10^–3 and 10^–4; virusinactivation occurred between 70° and 80°C.In electron micrographs the rod-shaped virus particles appeared similar to those of rattle virus.Virus could be detected in the roots of tobacco plants after the leaves had been inoculated with sap of diseasedAtropa-plants (Table 1). The reverse did not occur. Following immersion of the roots of tobacco plants in virus-containing sap these plants were potted in steamed soil. Subsequently the roots proved to be infected but the stems and leaves contained no virus. However,Atropa plants treated in the same way, did show leaf symptoms.It appeared, that the roots of young, healthy tobacco plants could become infected with virus, when grown in naturally infested soil for only tow days (Table 2). Fungus cultures isolated from diseased roots did not show any infectivity. Nematodes are probably the vectors of this virus (Sol, Van Heuvel & Seinhorst, 1960).Met medewerking van Merr.J. M. Dekhuyzen-Maasland, Dr. S. Gayed (Karthoum), Mej.C. van Heuven, C. de Vooys en Mej.R. van Wessem.     

Effects of surface and subsurface water application on nitrogen and sodium relations of desert graminoids of different geographic origin

This greenhouse study evaluated nitrogen and sodium relations of three desert graminoids (Distichlis spicata, Leymus triticoides, and Juncus arcticus) as affected by availability of surface water, subsurface water or both. These species are amply distributed in desert wetlands of western USA where surface and subsurface water are differentially available. Plants of the three species were collected from two areas of ecological distribution: Bishop, California and Burns, Oregon. Because nitrogen and sodium uptake by plants is highly linked to water availability we established three general hypotheses for this study: (1) nitrogen uptake would be greater when plants have surface water available, (2) sodium uptake would be greater when plants do not have surface water available, and (3) there are populations’ differences in the response of the species to water availability. We grew plants in two-layer pots in which soil water content in the upper and lower layers was controlled independently. The first hypothesis was partially supported as only Leymus triticoides seemed to preferentially depend on the top layer for nitrogen acquisition. With respect to the second hypothesis sodium concentration was indeed greatest when plants had no surface water, but only in D. spicata. The third hypothesis was also partially supported. The Oregon population of J. arcticus had 15% more nitrogen than the California population and the California population of D. spicata had 18% more sodium than the Oregon population. Our results underline the plant nutrient uptake implications of differential availability of water pools for common desert graminoid species.     

Nucleotide sequence and symptom modulating analysis of a Peanut stunt virus-associated satellite RNA from Poland: high level of sequence identity with the American PSV satellites

Two peanut stunt virus isolates from Poland (PSV-Ag and PSV-P) have been studied. The isolates produce similar systemic symptoms onNicotiana tabacum plants but the symptoms onN. benthamiana, Pisum sativum andDatura stramonium plants are much stronger for the PSV-P isolate. Analysis of the RNA extracted from purified virions by gel electrophoresis and RT-PCR amplification allowed the detection of a satellite RNA in the PSV-P isolate. The nucleotide sequence of this European PSV satellite was determined and found to have a high degree of identity with the sequences of the four American PSV satellites previously studied, which were found to have either no effect or ameliorate the PSV symptoms in tobacco plants (Collmer et al., 1985; Naidu et al., 1991). The possible role of the European PSV satellite in the modulation of viral symptoms has been studied but no effect was observed when the purified satellite was used with the PSV-Ag isolate as helper virus on any of the three hosts cited above.     

Occurrence of aphidborne viruses in southernmost South American populations of Fragaria chiloensis ssp. chiloensis

Wild and cultivated Fragaria chiloensis ssp. chiloensis (Fcc) plants were collected at different locations in southern Chile in order to determine the current viral status of this native strawberry. The following aphidborne viruses (ABVs): Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry crinkle virus (SCV) and Strawberry vein banding virus (SVBV), were found in wild and cultivated Fcc plants, but severe symptoms were not associated with viral infection. Furthermore, partial gene sequences of these ABV isolates were determined and displayed a high degree of conservation with virus isolates reported previously. In addition, partial gene sequences of SCV and SVBV from southernmost South American populations of Fcc are described for the first time. High‐throughput parallel sequencing (Illumina) of double‐stranded RNA was used to provide viral profiles of Fcc from different locations. Although strong evidence of novel viruses affecting Fcc was not found, it was confirmed that ABVs are the most frequent viruses affecting this subspecies. The information provided will help in the development of high‐quality molecular tools for virus detection and control in Fcc.     

Biological,serological and molecular characterisation of <Emphasis Type="Italic">Raspberry bushy dwarf virus</Emphasis> from grapevine and its detection in the nematode <Emphasis Type="Italic">Longidorus juvenilis</Emphasis>

In 2003, Raspberry bushy dwarf virus was found for the first time in grapevine. Since this was the first report of a non-Rubus natural host, information about it is sparse. During this study the grapevine isolates were characterised biologically, serologically and genetically and compared with known information about Rubus isolates. Infected plant material was used for mechanical inoculation of test plants, and for serological characterisation with monoclonal antibodies. Most of RNA 2 was sequenced and compared with other sequences from the database. Grapevine isolates infected Chenopodium murale systemically, which is not known for raspberry isolates. They were differentiated from Slovenian raspberry isolates with three monoclonal antibodies using TAS-ELISA. Phylogenetic analyses clustered grapevine isolates separately from raspberry isolates. Additionally, the virus was detected using nested RT-PCR in Longidorus juvenilis nematodes collected in an infected vineyard. Grapevine isolates of RBDV are distinct from raspberry isolates.     

Phytophthora species causing crown and root rot of tomato in southern Italy*

Phytophthora capsici, Phytophthora cryptogea and Phytophthora nicotianae were isolated from tomato plants with symptoms of crown and root rot in plastic‐house crops in Sicilia and Calabria (southern Italy). The species were identified primarily on the basis of morphological and cultural characteristics. The identification was confirmed using molecular methods, polyacrylamide gel electrophoresis (PAGE) of mycelial proteins and polymorphism of DNA sequences amplified by polymerase chain reaction using random primers (RAPD‐PCR). P. capsici caused significant losses in tomato crops that had succeeded capsicum crops. P. cryptogea was found to be the most frequent species causing basal stem rot of tomato, a disease of increasing importance in commercial tomato crops in plastic houses in Sicilia. P. nicotianae was common in plastic houses where poor drainage resulted in standing water.     

Isolation and partial characterization of polypeptides associated with phytotoxin in cultures of the cool-temperature biotype of Stemphylium botryosum pathogenic on alfalfa

Stemphylium botryosum pathogenic on alfalfa (Medicago sativa) produced a phytotoxin in a defined liquid medium which caused symptoms similar in sequence and appearance to stemphylium leafspot lesion development on attached alfalfa leaflets. The molecular weight of the phytotoxin was estimated to be 19500 by gel filtration chromatography. Treatment of partially purified phytotoxin with proteinase K or subtilisin resulted in loss of phytotoxic activity. Action of the proteases in inducing loss of phytotoxicity was inhibited by phenylmethylsulphonyl fluoride. Concentrated culture filtrates were fractionated by chromatofocusing, HPLC ion-exchange and gel filtration. Gel filtration also was followed by non-denaturing electrophoresis or isoelectric focusing (IEF). Analysis of sequential fractions from each procedure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that phytotoxic activity was consistently coincident with the presence of two major polypeptides (M_r = 26900 ± 2%; 19500 ± 4%) and a triplet of polypeptides (M_r = 15500). The constant association between the three polypeptides suggested an intermolecular interaction during fractionation. The phytotoxic activity eluted from the chromatofocusing column between pH 5·79 and 5·43 whereas IEF estimated the isoelectric point to be between 5·40 and 5·02.Immobilized concanavalin A and wheat germ lectin failed to bind phytotoxicity. Proteolytic activity in some phytotoxic gel filtration fractions was reduced by addition of phenyl-methylsulphonyl fluoride and separated from phytotoxicity by non-denaturing electrophoresis, indicating that phytotoxicity was not due to serine protease activity.Pathogenicity tests of the cool-temperature biotype on 20 plant species representing eight plant families resulted in a compatible interaction only on M. sativa and M. polymorpha. In contrast, sensitivity to the phytotoxin was observed on both pathogen-resistant and susceptible alfalfa clones along with all other plants tested with the exception of Triticum aestivum, Zea mays, Apium graveolens and Melilotus officinalis, indicating non-host-specificity of the phytotoxic polypeptides produced by the cool-temperature biotype.     

Further characterization of celery latent virus

Celery latent virus has been isolated from plants of celeriac (Apium graveolens var.rapaceum).Chenopodium amaranticolor andC. quinoa are good assay plants. Celery (A. graveolens var.dulce) and celeriac (13 cultivars tested) did not react with visible symptoms.Fourteen new artificial hosts were found. New systemic symptomless hosts areAnthriscus cerefolium, Nicotiana megalosiphon, Pisum sativum, Spinacia oleracea andTrifolium incarnatum. Systemic symptoms were caused in the pea cultivars Cicero and Dark Skin Perfection.Five aphid species, includingCavariella aegopodii, four of which not tested with the virus before, were unable to transmit the virus.Seed transmission was confirmed in celeriac (up to 34%) and inC. quinoa (up to 67%) and detected for the first time inAmaranthus caudatus. Detection is easier in seedlings and germinated seeds than in dry seeds.Results of purification were erratic but best at high pH (8 or 9). Sedimentation coefficient was 161 S.An antiserum reacted with purified virus in micro-precipitin tests (titer 256) but, especially at high salt concentrations also in agar gel (titer 64), presumably because of easy degradation of virus protein. Reactions in agar gel also occurred with crude extracts from virus-infectedC. quinoa and celeriac.With the electron microscope flexuous virus particles were found in low concentration in crude sap and in high concentration in purified preparations. Particle measurements revealed an average length of 885 nm (in one preparation 940 nm).Light microscopy did not show inclusion bodies in epidermal strips nor did electron microscopy of ultrathin sections reveal pinwheels and other structures typical of the potyvirus group. The virus evidently belongs to a new morphological group, possibly together with some other viruses hitherto insufficiently studied.The virus seems of potential economic importance only, but it is advised to use virus-free mother plants of celery for seed production.participating in the investigations from August 20th to December 19th 1972, supported by a fellowship of the Fundacion Juan March, Madrid, Spain.     

Multiplex RT-PCR for detection and identification of three necroviruses that infect olive trees

An optimized multiplex RT-PCR assay was developed to discriminate three necrovirus (Olive latent virus 1 (OLV-1), Tobacco necrosis virus D (TNV-D) and Olive mild mosaic virus (OMMV)) that infect olive trees. An olive orchard consisting of 54 trees of cv. “Galega vulgar” in the south of Portugal was surveyed. dsRNA fraction was used as template and revealed the 3 viruses, singly or in multiple infections, present in 17 out of 54 trees in the orchard. OMMV was the most frequent occurring in 15 trees, followed by OLV-1 in 12 and TNV-D in 4 plants. The results obtained showed that necrovirus- specific dsRNAs do exist in infected tissues in amounts below the resolution permitted by gel electrophoresis analysis and that the developed multiplex PCR based assay is of much higher sensitivity. The design of the specific primers described enabled, for the first time, to discriminate between OMMV and TNV-D by means of RT-PCR assays, an indispensable tool in identification, epidemiology and survey studies.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

Solanum dulcamara L. (Bitterzoet) als mogelijke bron voor bladrolvirus

Summary Dijkstra (1933) demonstrated thatSolanum dulcamara (Bittersweet, Woody Nightshade) can be a host plant of potato leaf-roll virus. It was, however, not known whether this wild growing plant in nature contains the virus. AsS. dulcamara is the only common perennial Solanacea in the Netherlands its role as a virus reseroir was investigated.At 25 localities plants ofS. dulcamara were collected, and tested for the presence of leaf-roll virus by transferring virus-freeMyzus persicae (Sulz.) after 24 hrs feeding to groups ofPhysalis floridana, where they fed for 24 hrs before being killed. All plants ofS. dulcamara appeared to contain leafroll virus, though they did not show symptoms. Also seedlings ofS. dulcamara from seed of a leafroll infested plant showed no symptoms before or after infection with leafroll bij transmission withMyzus persicae (Sulz.).The virus could be transmitted fromS. dulcamara to potato (var. Bintje) byMyzus persicae (Sulz.) and by graftingS. dulcamara on potato.Seedlings, grown from seed obtained from a plant which was shown to have leaf-roll, all appeared to be infected with leaf-roll. As so far no plants were found or obtained which had no leaf-roll virus, it is possible that inS. dulcamara leaf-roll virus is invariably transmitted with seed. But as the virus does not appear to be pathogenic inS. dulcamara, one might assume that what in potatoes and other plants is called leaf-roll virus, inS. dulcamara is an integral, natural component of the organism.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Identification and characterization of a mechanical transmissible begomovirus causing leaf curl on oriental melon

Oriental melon plants, Cucumis melo var. makuwa cv. Silver Light, showing virus-induced symptoms of mosaic, leaf curl and puckering were observed in the fields of eastern Taiwan in 2007. A virus culture, designated as SL-1, isolated from the diseased melon was established in systemic host plants, Nicotiana benthamiana and oriental melon, by mechanical inoculation. SL-1 did not react to the antisera against common cucurbit-infecting RNA viruses. Viral DNAs extracted from the diseased plant were amplified with the degenerate primers for begomoviruses. The full-length genomic DNA-A and DNA-B of SL-1 were sequenced and found to be closest, with 97.7% and 90.6% nucleotide identity, respectively, to Tomato leaf curl New Delhi begomovirus (ToLCNDV) cucumber isolate from a group of cucurbit-infecting begomoviruses. The virus SL-1 was designated as ToLCNDV oriental melon isolate (ToLCNDV-OM). The pathogenicity of ToLCNDV-OM was confirmed by agroinfection. Progeny virus from the agroinfected N. benthamiana plants was able to infect oriental melon by mechanical inoculation and caused symptoms similar to the original diseased melon in the field. The ToLCNDV-OM also infected five other species of cucurbitaceous plants by mechanical inoculation. This is the first report of a new ToLCNDV isolate causing severe disease on oriental melon in Taiwan.