Epidemiology of Raspberry Bushy Dwarf Virus in the Czech Republic

Extensive monitoring of the raspberry bushy dwarf virus (RBDV) in cultivated raspberry, wild raspberry and blackberry was conducted in 1994‐99. RBDV was revealed by ELISA in 31.6% of field samples, 15.7% plants in germplasm collections and in 43.8% of propagated plants. Infected cultivars were Aborigen, Balzam, Brigantina, Bulharský Rubín, Canby, Comox, F‐103, Findus, Gatineau, Glen Moy, Granát, Heritage, Lloyd George, M‐101, Mája, Meeker, Norfolk Giant, Norna, NS?‐1D‐101, Skeena, Trent, Veten, ZamatoS? and Zeva. The virus was detected in 6.5 and 6.7% of wild raspberry and wild blackberry plants, respectively, at 22.8% and 11.4% of sampled locations. Vegetatively propagated plants seem to be the main source for virus spread in cultivated raspberry, rather than naturally infested wild Rubus populations.     

Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses

Six blackberry or hybrid berry cultivars and 19 raspberry cultivars were assessed for their infectibility with, and sensitivity to, graft inoculation with 10 distinct viruses found infecting Rubus in the UK. Cultivars were grafted with each of, two isolates of the pollen borne raspberry bushy dwarf virus (RBDV), five aphid borne viruses: black raspberry necrosis, raspberry leaf mottle (RLMV), raspberry leaf spot (RLSV), rubus yellow net and raspberry vein chlorosis (RVCV); and isolates of the nematode transmitted nepoviruses, arabis mosaic, raspberry ringspot, strawberry latent ringspot and tomato black ring. All tested cultivars were infectible with a resistance breaking isolate of RBDV but only about half of that number with the Scottish type isolate of the virus. The raspberry cvs Autumn Bliss, and occasionally Glen Garry and Glen Prosen, developed leaf yellowing symptoms following infection with RBDV, but none of the other infected cultivars showed obvious leaf symptoms when kept in a heated glasshouse during the growing season. All tested cultivars were infectible with each of the four viruses transmitted in nature by the aphid, Amphorophora idaei. Most were infected symptomlessly, but seven cultivars developed severe leaf spotting symptoms due to infection with RLMV or RLSV. All but one of the raspberry cultivars were infectible with RVCV, which is transmitted in nature by the aphid Aphis idaei, and almost all infected plants developed leaf symptoms; only one of the hybrid berry or blackberry cultivars tested was infected with RVCV. In tests with the four nepoviruses, all tested cultivars, except Tummelberry, were infectible with at least one or more of these viruses. However, cultivars responded differently to challenge inoculation with different isolates of individual nepoviruses. Several cultivars developed chlorotic leaf mottling following infection with some nepovirus isolates. The implications of these results for virus control are discussed in the light of the changing pattern of virus and virus vector incidence in the UK.     

Molecular detection and characterisation of black raspberry necrosis virus and raspberry bushy dwarf virus isolates in wild raspberries

Virus‐derived small interfering RNAs (siRNAs) were extracted from leaves of wild raspberries (Rubus idaeus) sampled from three different regions in Finland and subjected to deep sequencing. Assembly of the siRNA reads to contigs and their comparison to sequences in databases revealed the presence of the bipartite positive‐sense single‐stranded RNA viruses, raspberry bushy dwarf virus (RBDV, genus Idaeovirus), and black raspberry necrosis virus (BRNV, family Secoviridae) in 19 and 26 samples, respectively, including 15 plants coinfected with both viruses. Coverage with siRNA reads [21 and 22 nucleotides (nt)] was higher in BRNV‐FI (Finland) RNA1 (79%) than RNA2 (45%). In RBDV, the coverage of siRNA reads was 89% and 90% for RNA1 and RNA2, respectively. Average depth of coverage was 1.6–4.9 for BRNV and 16.5–36.5 for RBDV. PCR primers designed for RBDV and BRNV based on the contigs were used for screening wild raspberry and a few cultivated raspberry samples from different regions. Furthermore, the sequences of BRNV RNA1 and RNA2 were determined by amplification and sequencing of overlapping contigs (length 1000–1200 nt) except for the 3′ and 5′ ends of RNA1 and RNA2 covered by primers. RNA1 of the Finnish BRNV isolate (BRNV‐FI) was 80% and 86% identical to BRNV‐NA (USA) and BRNV‐Alyth (UK), respectively, whereas the identity of NA and Alyth was 79%. RNA2 of BRNV‐FI was 84% and 80% identical to BRNV‐NA and BRNV‐Alyth, respectively, whereas NA and Alyth were 82% identical. Hence, the strains detected in Finland differ from those reported in the UK and USA. Our results reveal the presence of BRNV in Finland for the first time. The virus is common in wild raspberries and nearly identical isolates are found in cultivated raspberries as well. The results show that wild raspberries in Finland are commonly infected with RBDV or BRNV or both viruses and thus are likely to serve as reservoirs of RBDV and BRNV for cultivated Rubus spp.     

Isolates of raspberry bushy dwarf virus differing in Rubus host range

Isolates of raspberry bushy dwarf virus (RBDV) occurring in the field at East Mailing Research Station (EMRS), and an isolate from raspberry seed imported from the USSR, were found to differ from the Scottish type isolate (D200) of RBDV in that they infected red raspberry cultivars that are resistant, possibly immune, to isolate D200. Of several red raspberry, blackberry and hybrid berry cultivars and EMRS raspberry selections graft-inoculated with these recently discovered RBDV isolates only two raspberry cvs (Haida and Rannaya Sladkaya) and one EMRS selection did not become infected. Differences in the conclusions reached in two previous studies on the inheritance of resistance to RBDV in raspberry can be explained by the use of virus isolates that differed in Rubus host range.     

The occurrence and distribution of isolates of raspberry bushy dwarf virus in England

The distribution of distinct isolates of raspberry bushy dwarf virus (RBDV) in Rubus in England was studied. Isolates similar in Rubus host range to the Scottish type isolate (D200) were largely confined to the old red raspberry (Rubus idaeus) cv. Norfolk Giant, but were also encountered in a single plant of an unidentified raspberry cultivar and in a clump of wild R. idaeus. Outside East Mailing Research Station (EMRS) RBDV isolates with wider Rubus host ranges than that of the type isolate were found only and exclusively in hybrid berries (Loganberry, clones LY59 and L654, and Tayberry) in which infection ranged from < 1% to 100%. The significance of these findings is discussed.     

Unusual filamentous virus-like particles in symptomless blackberry associated with severe leaf curling in the virus indicator Rubus macraei, and anomalous data using a degenerate badnavirus primer in PCR of Rubus species

Electron microscopy of ultrathin sections of leaves of symptomless Himalaya Giant blackberry and of the virus indicator species, Rubus macraei, showing severe leaf curl symptoms following graft inoculation with scions from this blackberry, detected highly flexuous virus‐like particles with an unusual ‘beaded’ structure. Such particles were restricted to a few vascular cells and were distinct from P‐protein common in some such cells. This virus, provisionally named Hawaiian rubus leaf curl virus (HRLCV), symptomlessly infected a wide range of Rubus species and cultivars. Badnavirus‐like bacilliform particles were observed in some cells of a single R. macraei plant showing leaf curl symptoms following graft inoculation with the causal agent of this disease symptom from Himalaya Giant blackberry after passage through red raspberry, but not in any other material. PCR with primer sets for the badnaviruses Rubus yellow net virus and Gooseberry veinbanding associated virus, showed that no Rubus sources studied contained these viruses. However, using a sequence‐specific primer set designed from the sequence of the product generated with a badnavirus degenerate primer set, a specific product was amplified from healthy plants of all of 16 raspberry cultivars and two Rubus species, but not from 16 blackberry cultivars (including cv. Himalaya Giant). All of these sources were free from viruses known to occur in Rubus. Sequence analysis of this product showed no homology with any known badnavirus, or with any other published sequences. It seems most likely therefore that a region of the raspberry genome has been amplified using the degenerate badnavirus primer set and that it is absent from the blackberry genome.     

Natural infection with raspberry bushy dwarf virus (RBDV) of the putatively RBDV-resistant red raspberry cultivar Glen Moy, and the demonstration that it does not contain the RBDV resistance gene, Bu

When released to commerce in 1981, the red raspberry cv. Glen Moy was reported to be immune to the Scottish type isolate of raspberry bushy dwarf virus (RBDV-D200). Field observations of this cultivar in localities where RBDV was prevalent tended to support this claim of its resistance, but in the past 6–10 yr, RBDV infection has been reported in this cultivar in Australasia, USA and in several commercial crops in England. Therefore, experiments were made to investigate the reason(s) for this apparent anomaly using RBDV-infected material, putatively of cv. Glen Moy, from two locations in southern England and one each from Australia, New Zealand (NZ) and the USA. Genetic fingerprinting of genomic DNA from samples of these five RBDV-infected raspberry sources confirmed their identity as cv. Glen Moy. Comparisons of some serological and genomic properties of the five Glen Moy RBDV isolates indicated that, whilst they shared many properties with previously well characterised isolates of this virus, they were distinguishable from them. Characterisation of the isolate from NZ maintained in raspberry showed that it did not have a Rubus host range characteristic of resistance-breaking (RB) isolates, indicating that for this location, and probably also for those of Australia and the USA, RB isolates were not the cause of infection in cv. Glen Moy. When virus-tested plants of cv. Glen Moy and 45 progeny seedlings from the cross between cv. Glen Moy and the RBDV-susceptible cv. Autumn Bliss were graft inoculated with RBDV-D200, all grafted plants became infected indicating that cv. Glen Moy does not contain the RBDV resistance gene, Bu. Possible reasons for the previously reported resistance of cv. Glen Moy to RBDV are discussed.     

In vitro spore germination and infection of cultivars of Rubus and Rosa by downy mildews from both hosts

The cardinal temperatures for in vitro germination of conidia of imported and indigenous isolates of downy mildew from hosts in the genera Rubus and Rosa were similar. A high percentage of conidia germinated above 2°C and germination remained between 80% and 90% up to 15°C or 20°C, depending on the isolate. The highest incidence of disease on leaf disks of Tummelberry (blackberry × red raspberry) inoculated with an isolate of Peronospora rubi occurred at c. 15°C, with infection over a range from 2°C to 28°C. Tests on leaf disks in vitro, and leaflets of primocane and lateral shoots in plastic tunnels, with three hybrid berry (blackberry x red raspberry), six blackberry and nine red raspberry cultivars showed the hybrid berries to be most susceptible. In a plastic tunnel infected drupelets of red raspberry fruits developed more slowly and failed to ripen evenly compared with uninfected drupelets. Similar malformation of infected fruits occurred in a plantation of Tummelberry. An isolate of P. rubi attacked severely both Tummelberry and rose cv. Can Can. Fluorescence microscopy after staining with aniline blue showed that leaf disks of Tummelberry were extensively colonised by intercellular mycelium of P. sparsa isolated from rose, even though sporulation was sparse or absent. This supports the view that P. rubi and P. sparsa may be conspecific. Oospores of P. rubi were found routinely within leaf disks of Rubus cultivars inoculated in vitro and once in naturally infected leaflets of Tummelberry.     

The genome of black raspberry (Rubus occidentalis)

Black raspberry (Rubus occidentalis) is an important specialty fruit crop in the US Pacific Northwest that can hybridize with the globally commercialized red raspberry (R. idaeus). Here we report a 243 Mb draft genome of black raspberry that will serve as a useful reference for the Rosaceae and Rubus fruit crops (raspberry, blackberry, and their hybrids). The black raspberry genome is largely collinear to the diploid woodland strawberry (Fragaria vesca) with a conserved karyotype and few notable structural rearrangements. Centromeric satellite repeats are widely dispersed across the black raspberry genome, in contrast to the tight association with the centromere observed in most plants. Among the 28 005 predicted protein‐coding genes, we identified 290 very recent small‐scale gene duplicates enriched for sugar metabolism, fruit development, and anthocyanin related genes which may be related to key agronomic traits during black raspberry domestication. This contrasts patterns of recent duplications in the wild woodland strawberry F. vesca, which show no patterns of enrichment, suggesting gene duplications contributed to domestication traits. Expression profiles from a fruit ripening series and roots exposed to Verticillium dahliae shed insight into fruit development and disease response, respectively. The resources presented here will expedite the development of improved black and red raspberry, blackberry and other Rubus cultivars.     

Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids

Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi‐persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80–150 × 25–30 nm, similar to those of badnaviruses. A1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR‐amplified viral DNA fragment, RYNV‐specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus‐like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV‐infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field‐grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV‐infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes.     

Genomic characterisation and the detection of raspberry chromatin in polyploid Rubus

 This paper reports genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) data for chromosomes of raspberry (Rubus idaeus 2n=2x=14), blackberry (Rubus aggregate, subgenus Eubatus. 2n=2–12x=14–84) and their allopolyploid derivatives used in fruit breeding programmes. GISH was used to discriminate labelled chromosomes of raspberry origin from those of blackberry origin in allopolyploid hybrid plants. The raspberry chromosomes were labelled by GISH at their centromeres, and 1 chromosome was also labelled over the short arm. In one allopentaploid plant a chromosome carried a terminal signal. Karyotype analysis indicated that this is a blackberry chromosome carrying a raspberry translocation. GISH analysis of an aneuoctaploid blackberry cv ‘Aurora’ (2n=8x=58) showed that both whole and translocated raspberry chromosomes were present. The basic Rubus genome has one ribosomal DNA (rDNA) locus, and in all but one case all levels of ploidy had the expected multiples of rDNA loci. Interestingly, in the blackberry cv ‘Aurora’, there were only six sites, two less than might be predicted from its aneuoctaploid chromosome number. Our results highlight the potential of GISH and FISH for genomic designation, physical mapping and introgression studies in Rosaceous fruit crops. Received: 20 February 1998 / Accepted: 12 May 1998     

香蕉束顶病毒基因克隆和序列分析

对香蕉束顶病毒（ＢＢＴＶ）中国分离株ＤＮＡ组份Ｉ（ＤＮＡ－１）、外壳蛋白（ＣＰ）和运转蛋白（ＭＰ）基因进行了克隆和序列分析。ＢＢＴＶＤＮＡ－１含有１１０３个核苷酸,与南太平洋和亚洲分离株分别有８７％－８８％ ９６．９－９８％的核苷酸序列同源性。由ＤＮＡ－１编码的复制酶含有１８６个在酸残基。与南太平洋和亚洲分离株分别有８４．４％－９５．８％和９７．６％、９８．０％的氨基酸序列同源性。外壳蛋白基因由５     

Mycoplasma-like bodies associated with Rubus stunt disease

Mycoplasma-like bodies were found by electron microscopy in the sieve tubes of blackberry, loganberry and raspberry plants showing symptoms of Rubus stunt disease, but not in those of normal plants. Watering diseased blackberry plants with aureomycin caused remission of the symptoms and greatly decreased the number of mycoplasma-like bodies.     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Low Genetic Diversity of the Coat Protein Gene among Blueberry Red Ringspot Virus Isolates

Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.     

Development and bin mapping of strawberry genic-SSRs in diploid <Emphasis Type="Italic">Fragaria</Emphasis> and their transferability across the Rosoideae subfamily

Cultivated strawberry (Fragaria × ananassa) together with other economically important genera such as Rosa (roses) and Rubus (raspberry and blackberry) belongs to the subfamily Rosoideae. There is increasing interest in the development of transferable markers to allow genome comparisons within the Rosaceae family. In this report, 122 new genic microsatellite (SSR) markers have been developed from cultivated strawberry and its diploid ancestor Fragaria vesca. More than 77% of the sequences from which the markers were developed show significant homology to known or predicted proteins and more than 92% were polymorphic among strawberry cultivars, representing valuable markers in transcribed regions of the genome. Sixty-three SSRs were polymorphic in the diploid Fragaria reference population and were bin-mapped together with another five previously reported but unmapped markers. In total, 72 loci were distributed across the seven linkage groups. In addition, the transferability of 174 Fragaria SSRs to the related Rosa and Rubus genera was investigated, ranging from 28.7% for genic-SSRs in rose to 16.1% for genomic-SSRs in raspberry. Among these markers, 33 and 16 were both localized in the diploid Fragaria reference map and cross-amplified in rose and raspberry, respectively. These results indicate that transferability of SSRs across the Rosoideae subfamily is limited. However, we have identified a set of Fragaria markers, polymorphic in the diploid reference population, which cross-amplified in both Rosa and Rubus, which represents a valuable tool for comparative mapping and genetic diversity analyses within the Rosoideae subfamily.     

Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease

After graft inoculation with rubus yellow net virus (RYNV), 12 of 34 Rubus species and cultivars developed noticeable symptoms. R. macraei developed the most conspicuous symptoms and is recommended as an improved indicator plant. In attempts to determine the cause of raspberry veinbanding mosaic, a disease in which RYNV is involved, several European and North American red raspberry cvs were graft-inoculated with RYNV and three other aphid-borne viruses, black raspberry necrosis (BRNV), raspberry leaf mottle (RLMV) and raspberry leaf spot, singly and in all combinations. In periods of up to 4 yr, classical veinbanding mosaic symptoms developed in sensitive cvs only when they contained both RYNV and RLMV. These symptoms were intensified in plants co-infected with additional viruses. Veinbanding mosaic disease did not develop in any of 11 cvs infected with RYNV + BRNV, the combination of viruses previously assumed to be responsible for this disease in Britain and North America.     

Genetic diversity and population structure of Sorghum mosaic virus infecting Saccharum spp. hybrids

Sugarcane mosaic disease is widespread in many countries and has been identified to be caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV). Viral surveys of SCMV, SrMV and SCSMV were performed from 104 leaf samples of Saccharum spp. hybrid growing in China and two leaf samples in Myanmar. Sorghum mosaic virus was a major causal agent for sugarcane mosaic disease in China whereby 72.1% (75/104) of samples had SrMV infection alone, 6.7% (7/104) were mixed with SCMV and 17.3% (18/104) were mixed with SCSMV. Sugarcane streak mosaic virus infection alone occurred in 3.8% (4/104) of samples, but no single infections were observed for SCMV. Two viruses (SrMV and SCSMV) were detected in sugarcane mosaic samples in Myanmar. Phylogenetic analysis revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes based on the CP sequences (825 nucleotides) of 113 Chinese and 2 Burmese isolates from this study and 73 isolates reported worldwide. Six clearly distinct SrMV phylogroups (G1–G6) were formed and shared 74.3–94.1% nucleotide identity and 84.7–98.1% amino acid identity of CP sequences. SrMV‐G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces. The unique SrMV‐G6 phylogroup only occurred in Yunnan province. Insertion/deletion mutations, negative selection and frequent gene flow are factors driving the genetic evolution and population structure of SrMV in China.     

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019–2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96–99.5% nucleotide and 94.40–99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li’s F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.