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.     

Genetic control of the reactions of raspberry to black raspberry necrosis, raspberry leaf mottle and raspberry leaf spot viruses

Black raspberry necrosis virus (BRNV) induces a severe apical necrosis in black raspberry (Rubus occidentalis) but fails to induce diagnostic symptoms in red raspberry. However, BRNV infection of F₁, F₂ and F₃ hybrids from the cross black raspberry × red raspberry induced mosaic symptoms of varying intensity but no typical apical necrosis. In a survey of 28 red raspberry cultivars, a few developed severe angular chlorotic leaf spots when infected with raspberry leaf mottle virus and a few others did so when infected with raspberry leaf spot virus. These reactions were determined by single dominant genes designated Lm and Ls respectively. The value of the different host reactions for controlling the effects and spread of these viruses is discussed.     

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.     

Studies on the inheritance in the red raspberry of immunities from three nematode-borne viruses

Lethal or chronic diseases of the raspberry caused by the nematodeborne viruses raspberry ringspot, arabis mosaic and tomato black ring can cause serious reductions in the productivity of raspberry plantations, but the existence of clear-cut immunities from these diseases provides a basis for control through plant breeding. The inheritance of these immunities was studied by means of graft tests on families of raspberry seedlings. Immunity from each virus was found to be dominant to susceptibility, but there was evidence that more than one gene was concerned in each case: while it was not possible to decide whether the second gene was a dominant complementary or a linked recessive affecting the viability of the immune segregates, the frequent occurrence in the raspberry of aberrant segregation ratios due to such lethal genes makes the latter explanation the more probable. There was also evidence of linkage between the genes for the three immunities. The experiment confirmed the practicability of breeding to incorporate genes for immunities from these three viruses into new raspberry varieties.     

Biology of the European large raspberry aphid (Amphorophora idaei): its role in virus transmission and resistance breakdown in red raspberry

1 The European large raspberry aphid Amphorophora idaei Börner is the most important vector of viral diseases afflicting commercially grown red raspberry ( Rubus idaeus L.) in Northern Europe, with European raspberry production amounting to 416 000 tonnes per annum. This review synthesizes existing knowledge on its biology and interactions with other organisms, including its host plant and the viral pathogens it vectors.
2 Information about trophic interactions with other insect herbivores and natural enemies is reviewed. Vine weevils Otiorhynchus sulcatus compromise aphid resistance in some raspberry cultivars, increasing A.   idaei abundance by 80%. Parasitoids show mixed success in parasitizing A.   idaei , although Aphidius ervi attack rates more than doubled when A.   idaei fed on a partially susceptible raspberry cultivar, compared with a resistant variety. These findings are discussed in the context of potential biological control as part of an integrated pest and disease management framework.
3  Amphorophora idaei transmits four known viruses: Black raspberry necrosis virus, Raspberry leaf mottle virus, Raspberry leaf spot virus and Rubus yellow net virus , with A.   idaei taking as little as 2 min to transmit some viruses.
4 Existing control strategies, including resistant cultivars, insecticides and eradication of disease from parent plants, are described. In particular, strong selection pressures have resulted in A .  idaei overcoming genetic resistance in many raspberry cultivars and most insecticides are now ineffective.
5 Future directions for the sustained control of A.   idaei are suggested, taking into consideration the possible effects of climate change and also changes in agronomic practices in U.K. agriculture.     

Further studies on the occurrence and inheritance of resistance in red raspberry to a resistance-breaking strain of raspberry bushy dwarf virus

A strain of raspberry bushy dwarf virus (RBDV-RB), discovered in England in 1981, readily infects by grafting many raspberry cvs that have gene Bu, which confers strong resistance or immunity to isolates of the common strain. Haida is one of two cultivars that are highly resistant or immune from RBDV-RB, but both its parents, cvs Creston and Malling Promise, are infectible. Studies of the segregation of resistance to both RBDV-RB and a common strain of RBDV (D200) in four progenies related to cv. Haida or its two parents, showed that resistance to RBDV-RB was heritable and occurred when gene Bu was present with a second resistance component whose inheritance is probably multigenic. There was some indication that the second component might be a form of partial resistance to graft inoculation of varying expression, and that cv. Haida possesses this resistance at a high level that has not been distinguished from immunity in the graft inoculations used. Cultivars Creston and Malling Promise possibly have this resistance to a lesser degree, while resistance in cv. Heritage has been distinguished from immunity only by extensive graft tests. Some possible implications for breeding RBDV-RB resistant cultivars are discussed.     

Association of raspberry bushy dwarf virus with raspberry yellows disease; reaction of Rubus species and cultivars, and the inheritance of resistance.

The agent of raspberry yellows disease is transmitted by grafting but not by aphids and is resistant to thermotherapy. Further studies showed that it is transmitted by inoculation of sap through seed; it is probably transmitted to plants by pollination. Raspberry bushy dwarf virus (RBDV) shares all these attributes and is known to infect all yellows-sensitive raspberry cultivars except Puyallup and Sumner; however, neither of these cultivars has been tested by graft inoculation with RBDV. RBDV commonly infects plants symptomiessly, even those of yellows-sensitive cultivars, but it induced yellows when inoculated either manually to Norfolk Giant raspberry or by grafting to a yellows-sensitive raspberry selection. The evidence suggests that RBDV is the causal agent of yellows disease but that symptom expression is greatiy dependent on genetic and environmental factors. Many red raspberry cultivars are resistant, probably immune, to the type culture of RBDV and this character was shown to be conferred by a single dominant gene designated Bu.     

Spread of raspberry bushy dwarf virus by pollination, its association with crumbly fruit, and problems of control

Raspberry bushy dwarf virus (RBDV) was transmitted to raspberry seed both through the pollen and through the ovule and it infected plants pollinated with infected pollen. It did not infect plants prevented from flowering, and transmission through pollen seems to be the only method of spread in the field; in the proximity of infectors, most plants became infected during the first two or three flowering seasons. Plants containing RBDV showed no obvious symptoms, but healthy or infected flowers pollinated with infected pollen produced ‘crumbly’ fruit, containing a high proportion of aborted drupelets. RBDV was difficult to eliminate from infected raspberry by heat therapy. Raspberry cultivars that fail to become infected naturally were also immune to infection by grafting. Use of immune cultivars offers the only method of control and, because infected plants may produce crumbly fruit, future cultivars should if possible possess immunity to RBDV.     

Further properties of black raspberry latent virus, and evidence for its relationship to tobacco streak virus

Purified preparations of an isolate of black raspberry latent virus (BRLV) contained quasispherical particles with a mean diameter of 28·5 nm; these particles were resolved into three sedimenting components (s_{20, w}= 82S, 95S and 104S), but when centrifuged to equilibrium in caesium chloride solution they formed a single infective band (σ= 1·35 g/cm³). During electrophoresis in polyacrylamide gels, virus particles separated into three classes, and virus RNA was resolved into three major (mol. wt 1·35, 1·10 and 0·85 × 10⁶) and one minor (mol. wt 0·4 × 10⁶) component. The protein from virus particles had an estimated mol. wt of 28000. Isolates of BRLV were found to be serologically related but not identical to some strains of tobacco streak virus. No symptoms developed in black raspberry seedlings infected with BRLV by mechanical inoculation, nor in eight red raspberry cultivars infected by graft inoculation. However, graft inoculation of BRLV to Rubus henryi, R. phoenicolasius and Himalaya blackberry induced symptoms typical of necrotic shock disease.     

Rubus host range, symptomatology and ultrastructural effects of a British isolate of black raspberry necrosis virus

Scions from red raspberry (Rubus idaeus) plants naturally infected with an aphid- and sap-transmissible virus, code-named 52V, always induced apical necrosis in R. occidentals signifying the presence of black raspberry necrosis virus (BRNV), whereas plants free from 52V did not. These and other findings provide strong circumstantial evidence that 52V is an isolate of BRNV, the heat-labile member of the pair of viruses that together cause raspberry veinbanding mosaic disease. On grafting with R. idaeus scions containing a 52V culture of BRNV free from other detectable viruses, all of twenty-two red raspberry cultivars and four other Rubus species were infected symptomlessly but apical necrosis developed in R. henryi and R. molaccanus. Electron microscopy of thin sections of 52V-infected Chenopodium quinoa, R. henryii and R. occidentalis showed areas of dead cells in the vascular tissue and leaf blade. Some of the cells adjacent to these areas had cell wall outgrowths and many of the plasmodesmata contained virus-like particles c. 25 nm in diameter arranged in a single file.     

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.     

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.     

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.     

Association of different kinds of bacilliform particle with vein chlorosis and mosaic diseases of raspberry (Rubus idaeus)

Thin sections of diseased raspberry (Rubus idaeus) were examined by electron microscopy. Plants of the cv. Baumforth's B and of an aphid (Amphorophora rubi)-resistant breeding selection (6820/54), both infected with raspberry vein chlorosis virus (RVCV) but not with other detectable viruses, contained large bacilliform particles c. 430 × 65 nm. Particles occurred in the cytoplasm and perinuclear space of a small proportion of xylem parenchyma cells. They had an inner core c. 25–30 nm in diameter with cross-banding of periodicity 4·5 nm, and were bounded by an outer membrane. They are probably the particles of RVCV. Plants of cv. Mailing Jewel and of a selection (M14) both showing symptoms of raspberry mosaic (veinbanding) disease contained smaller bacilliform particles c. 125 × 30 nm, which occurred singly or in clusters in the cytoplasm of a small proportion of vascular parenchyma cells. It is not known which, if any, of the viruses associated with raspberry mosaic are represented by the particles.     

Propagation of black raspberry necrosis virus (BRNV) in mixed culture with solanum nodiflorum mottle virus, and the production and use of BRNV antiserum

The concentration of particles of black raspberry necrosis virus (BRNV), which is normally extremely low in herbaceous plants, increased about 1000-fold when Nicotiana clevelandii plants were inoculated with a mixture of BRNV and an unrelated virus, solanum nodiflorum mottle (SNMV). In sap from N. clevelandii infected with the mixed culture, BRNV infectivity survived dilution to 10?⁴ but not 10?⁵, and storage for 6 but not 8 days at 20 ^oC, for 6 but usually not 10 days at 4 ^oC and for more than 13 days at – 15 ^oC. When plants were inoculated with the mixed culture, BRNV induced typical symptoms in several Chenopodium species and infected several previously unreported hosts. Purified preparations of particles of the mixed culture contained only a small proportion of BRNV particles, which sedimented in sucrose density gradients as two components, one, probably non-infective, of c. 505, and the other, infective, of 120-130S. An antiserum prepared to purified particles of the mixed culture was cross-absorbed with SNMV particles and used in indirect ELISA to detect BRNV in herbaceous plants infected with the mixed culture, and also in a wide range of Rubus species, cultivars and hybrids infected naturally, by grafting or by inoculation with the aphid Amphorophora idaei. The reliability of ELISA for detecting BRNV in raspberry leaves depended on the cultivar and time of year. Some cultivars, such as Glen Clova, had low concentrations of BRNV, which was detected reliably only in late spring/early summer, whereas other cultivars, such as Lloyd George and Mailing Enterprise, had greater BRNV concentrations. In small-scale surveys in eastern Scotland, BRNV was detected by ELISA in many raspberry cvs, including some that contain major gene resistance to the vector, A. idaei; in five of nine raspberry stocks entered for the Standard grade certificate but in none of five stocks entered for the Stock Cane certificate; and in 40% of wild raspberry and 14% of wild bramble plants growing near commercial raspberry crops. The significance of these findings for the control of BRNV is discussed.     

Resistance to Didymella applanata in red raspberry and some related species

When raspberry canes were inoculated in summer with a mycelial inoculum of Didymella applanata resistant genotypes developed relatively small lesions which produced few fruiting bodies in the following spring. Scores of the frequency of these fruiting bodies provided the best discrimination between genotypes. Very strong resistance was found in R. pileatus, R. occidentalis and R. coreanus and in hybrids of these species with red raspberry. Hybrids with R. crataegifolius were less resistant. Resistance was also found in Malling 1473/35, a derivative of red raspberry cv. Chief, and in red raspberry segregates with gene H which determines cane pubescence. Both the expression of resistance and the ranking of resistance sources for their resistance was similar for D. applanata to that reported for Botrytis cinerea.     

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.     

Location of the mechanism of resistance to Amphorophora agathonica (Hemiptera: Aphididae) in red raspberry

The aphid Amphorophora agathonica Hottes (Hemiptera: Aphididae) is an important virus vector in red (Rubus idaeus L.) and black (Rubus occidentalis L.) raspberries in North America. Raspberry resistance to A. agathonica in the form of a single dominant gene named Ag1 has been relied upon to help control aphid-transmitted plant viruses; however, the mechanism of resistance to the insect is poorly understood. Aphid feeding was monitored using an electrical penetration graph on the resistant red raspberry 'Tulameen' and compared with a susceptible control, 'Vintage'. There were no differences in pathway feeding behaviors of aphids as they moved toward the phloem. Once in the phloem, however, aphids feeding on resistant plants spent significantly more time salivating than on susceptible plants, and ingested significantly less phloem sap. This suggests that a mechanism for resistance to A. agathonica is located in the phloem. Reduced ingestion of phloem may result in inefficient acquisition of viruses and is a likely explanation for the lack of aphid-transmitted viruses in plantings of resistant cultivars.     

Ripening-related changes in raspberry cell wall composition and structure

Cell walls were prepared from the fruit of two cultivars of raspberry at three stages of ripening; green, white and red (ripe). The cultivars. Glen Clova and Glen Prosen, are subjectively classified, at harvest by growers, as soft and firm fruit, respectively. The cell walls were analysed for neutral sugar composition, uronic acid content, degree of methyl esterification, lignin and ferulic acid-derived dehydrodimers. Solid-state 31C NMR and diffuse reflectance infrared (DRIFT) spectra were acquired for the cell wall residues. For both cultivars the progression from green to white produced minimal changes, save for a reduction in pectin. NMR analyses indicated that the solubilized pectin was acetylated. Progression to the red (ripe) stage, in both cultivars, was accompanied by a reduction in the ordered cellulose and a dramatic reduction in pectin content and the degree of methyl-esterification. Significantly, the softer fruit (Glen Clova) exhibited greater reductions in both parameters, implicating increased pectin hydrolysis, as one of the main factors contributing to the difference in firmness between the cultivars. A relative increase in cell wall-associated protein was seen at the red stage. The nature and function of the protein(s) are, as yet unknown.     

The effect of resistance to Amphorophora rubi in raspberry (Rubus idaeus) on the spread of aphid-borne viruses

The effectiveness of resistance to the aphid Amphorophora rubi in restricting the spread of aphid-borne viruses was assessed in a field experiment using six genotypes of red raspberry. In one block of the experiment, the genotypes alternated with rows of virus-infected Mailing Jewel raspberry, and in the other they alternated with virus-free Mailing Jewel. During 4 years, the numbers of A. rubi and the amount of 52V virus spread in the two blocks were similar, suggesting that this virus was mostly introduced from outside the plots. Lloyd George and Mailing Jewel raspberry became heavily infested with A. rubi and were rapidly infected with raspberry leaf mottle, raspberry leaf spot and 52V viruses. Glen Clova and Norfolk Giant raspberry, which contain minor genes for resistance to A. rubi, were infested with fewer A. rubi and virus spread more slowly in these cultivars. A. rubi were rare on Mailing Orion and an East Mailing raspberry selection (888/49) which have genes A₁ and A₁₀ respectively for resistance to A.rubi, and these plants remained largely free of virus. The role of minor and major gene resistance to A. rubi in restricting virus spread is discussed. A few Macrosiphum euphorbiae and Myzus ornatus were recorded on several of the raspberry genotypes.