Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Polyribosomes from aging apple and cherry fruit

The sequence of events which occurs during the ripening of the Passe-Crassane pear fruit have been previously studied. In this work, we have investigated the ripening of another climacteric fruit (Pyrus malus L. cv Golden Delicious) and of a nonclimacteric fruit (Prunus avium L. cv Bigarreau Napoléon). We show that both climacteric fruits exhibit the same preclimacteric sequence of events. Differences exist, however, between the Golden Delicious apple and the Passe-Crassane pear in that the protein synthesis capacity of the two fruits is not the same during the over-ripening period. On the other hand, a nonclimacteric fruit, the Bigarreau Napoléon cherry, does not show an increase in its protein synthesis capacity during the over-ripening period.     

Efficient genetic transformation of red raspberry,Rubus ideaus L.

We have developed an efficient transformation system for red raspberry (Rubus ideaus L.) using Agrobacterium mediated gene transfer. Using this system we have successfully introduced a gene that encodes an enzyme, S-adenosylmethionine hydrolase (SAMase), in raspberry cultivars Meeker (MK), Chilliwack (CH) and Canby (CY). Leaf and petiole expiants were inoculated with disarmed Agrobacterium tumefaciens strain EHA 105 carrying either of two binary vectors, pAG1452 or pAG1552, encoding gene sequences for SAMase under the control of the wound and fruit specific tomato E4 promoter. Primary shoot regenerants on selection medium were chimeral containing both transformed and non-transformed cells. Non-chimeral transgenic clones were developed by iterative culture of petiole, node and leaf explants, on selection medium, from successive generations of shoots derived from the primary regenerants. Percent recovery of transformants was higher with the selection marker gene hygromycin phosphotransferase (hpt), than with neomycin phosphotransferase (nptII). Transformation frequencies of 49.6%, 0.9% and 8.1% were obtained in cultivars Meeker, Chilliwack and Canby respectively from petiole expiants using hygromycin selection. Genomic integration of transgenes was confirmed by Southern hybridization. Transgenic plants from a total of 218 independent transformation events (161 MK, 4 CH, 53 CY) have been successfully established in soil.Abbreviations ACCO amincocyclopropane-1-carboxylic acid oxidase - AS acetosyringone - BA 6-benzylaminopurine - CH cultivar Chilliwack - CY cultivar Canby - cv cultivar - hpt hygromycin phosphotransferase - IBA indolebutyric acid - MK cultivar Meeker - npt II neomycin phosphotransferase - SAMase S-adenosylmethionine hydrolase - TDZ Thidiazuron (N-phenyl-N'-l,2,3-thidiazol-5-ylurea)     

Neuraminidase deficiency in the cherry red spot-myoclonus syndrome

Two patients with the cherry red spot-myoclonus syndrome excreted excessive quantities of sialylated oligosaccharides in urine. Skin fibroblasts from both patients had a severe deficiency of neuraminidase activity using α-L-N-acetylneuraminosyl-(2→6′) lactose and a sialylhexasaccharide from their urine as substrates. A less severe deficiency was found using 2-(3′ methoxyphenyl)-N-acetylneuraminic acid, fetuin, and α-L-N-acetylneuraminosyl-(2→3′) lactose as substrates. I propose that the primary enzyme defect in this autosomal recessive disorder is a deficiency of lysosomal neuraminidase.     

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.     

Construction of black (Rubus occidentalis) and red (R. idaeus) raspberry linkage maps and their comparison to the genomes of strawberry, apple, and peach

The genus Rubus belongs to the Rosaceae and is comprised of 600-800 species distributed world-wide. To date, genetic maps of the genus consist largely of non-transferable markers such as amplified fragment length polymorphisms. An F(1) population developed from a cross between an advanced breeding selection of Rubus occidentalis (96395S1) and R. idaeus 'Latham' was used to construct a new genetic map consisting of DNA sequence-based markers. The genetic linkage maps presented here are constructed of 131 markers on at least one of the two parental maps. The majority of the markers are orthologous, including 14 Rosaceae conserved orthologous set markers, and 60 new gene-based markers developed for raspberry. Thirty-four published raspberry simple sequence repeat markers were used to align the new maps to published raspberry maps. The 96395S1 genetic map consists of six linkage groups (LG) and covers 309 cM with an average of 10 cM between markers; the 'Latham' genetic map consists of seven LG and covers 561 cM with an average of 5 cM between markers. We used BLAST analysis to align the orthologous sequences used to design primer pairs for Rubus genetic mapping with the genome sequences of Fragaria vesca 'Hawaii 4', Malus × domestica 'Golden Delicious', and Prunus 'Lovell'. The alignment of the orthologous markers designed here suggests that the genomes of Rubus and Fragaria have a high degree of synteny and that synteny decreases with phylogenetic distance. Our results give unprecedented insights into the genome evolution of raspberry from the putative ancestral genome of the single ancestor common to Rosaceae.     

A strategy for developing representative germplasm sets for systematic QTL validation,demonstrated for apple,peach, and sweet cherry

Horticultural crop improvement would benefit from a standardized, systematic, and statistically robust procedure for validating quantitative trait loci (QTLs) in germplasm relevant to breeding programs. Here, we describe and demonstrate a strategy for developing reference germplasm sets of perennial, clonally propagated crops, especially those with long juvenile periods. Germplasm is chosen to efficiently represent important members of larger pedigree-connected genepools. To facilitate validation of multiple QTLs, genome-wide representation of alleles is optimized for designated important breeding parents (IBPs) by estimating average allelic representation in relatives. The strategy and arising principles were demonstrated in a simulated germplasm set. Strong statistical power can be achieved with a carefully chosen germplasm set composed of IBPs, their numerous unselected progenies and close relatives, and all available founders and intermediate ancestors. Crop Reference Sets were developed in the marker-assisted breeding (MAB)-enabling “RosBREED” project as a base resource for QTL validation in US breeding germplasm of apple (Malus × domestica), peach (Prunus persica), and sweet cherry (Prunus avium) consisting of 467, 452, and 268 individuals, respectively. These sets adequately represent the most designated IBPs, have distinct advantages for QTL validation over other germplasm arrangements of equal size, and are recommended as a base resource for QTL validation by breeders of these US crops. The strategy described here can be used to develop efficient reference germplasm sets suiting other breeding genepools or to calculate the statistical power for QTL validation of germplasm sets already established.     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

New insights into the bioavailability of red raspberry anthocyanins and ellagitannins

Red raspberries, containing ellagitannins and cyanidin-based anthocyanins, were fed to volunteers and metabolites appearing in plasma and urine were analysed by UHPLC-MS. Anthocyanins were not absorbed to any extent with sub nmol/L concentrations of cyanidin-3-O-glucoside and a cyanidin-O-glucuronide appearing transiently in plasma. Anthocyanins excreted in urine corresponded to 0.007% of intake. More substantial amounts of phase II metabolites of ferulic acid and isoferulic acid, along with 4′-hydroxyhippuric acid, potentially originating from pH-mediated degradation of cyanidin in the proximal gastrointestinal tract, appeared in urine and also plasma where peak concentrations were attained 1–1.5 h after raspberry intake. Excretion of 18 anthocyanin-derived metabolites corresponded to 15.0% of intake, a figure substantially higher than obtained in other anthocyanin feeding studies. Ellagitannins pass from the small to the large intestine where the colonic microbiota mediate their conversion to urolithins A and B which appeared in plasma and were excreted almost exclusively as sulfate and glucuronide metabolites. The urolithin metabolites persisted in the circulatory system and were excreted in urine for much longer periods of time than the anthocyanin metabolites although their overall urinary recovery was lower at 7.0% of intake. It is events originating in the proximal and distal gastrointestinal tract, and subsequent phase II metabolism, that play an important role in the bioavailability of both anthocyanins and ellagitannins and it is their metabolites which appear in the circulatory system, that are key to elucidating the mode of action(s) underlying the protective effects of these compounds on human health.     

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.     

Resistance to Leptosphaeria coniothyrium in the red raspberry and some related species

Useful resistance to Leptosphaeria coniothyrium was found in the red raspberry cv. Latham, but its effectiveness was affected by the stage of growth reached by the canes when they were inoculated and by the kind of inoculum used: it was effective when field-grown canes were inoculated in mid-August but less so when they were inoculated in early July, and more effective against a spore inoculum than against a mycelial one. Resistance was inherited as a dominant multigenic character, but there were interactions between parents and two very favourable combinations were found.
The species Rubus pileatus, R. mesogaeus, R. coreanus and R. lasiocarpus showed resistance when inoculated in pot experiments, but F₁ hybrids obtained by crossing them with the raspberry were largely susceptible. The problems of using these resistance sources in breeding are discussed.     

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.     

Benefits of ethylene removal during apple storage

The potential use of an ethylene absorbent in controlled atmosphere storage of two varieties of apple has been investigated. With Golden Delicious, the rise in ethylene concentration during controlled atmosphere storage can be delayed for about 40 days but not prevented by inclusion of potassium permanganate in the storage container. But with Bramley's Seedling, potassium permanganate can delay ethylene accumulation for over 200 days. Ethylene treatment of Bramley's Seedling in a flowing stream of 5% CO₂:3% O₂:92% N₂ caused accelerated softening, accumulation of α-farnesene and earlier onset of superficial scald. Use of potassium permanganate to remove ethylene during storage in static controlled atmosphere conditions retarded all three processes in Bramley's Seedling kept in 5% CO₂:3% O₂ and in 9% CO₂:12% O₂. However, in one experiment, ethylene removal in 5% CO₂:3% O₂, led to external and internal symptoms of CO₂ damage. A subsequent investigation of the combined effects of harvest date, store temperature and ethylene removal in 5% CO₂:3% O₂ did not show any damage or accumulation of succinic acid which is known to be involved in CO₂ injury. This experiment revealed that ethylene removal could be successfully accomplished on Bramley fruit harvested up to a month after the usual date and little α-farnesene accumulated in this fruit. Nevertheless scald did develop on late picked fruit and this raises doubts about the causal role of farnesene in scald.     

Effect of machine-harvester wounds and Leptosphaeria coniothyrium on yield of red raspberry

When wounds on raspberry canes caused by machine harvesting in the previous year were infected by Leptosphaeria coniothyrium the potential yield per cane was reduced by 45% compared with that of uninfected hand harvested canes. However, the corresponding reduction per plot was less (c. 25%) because the yield of wounded but uninfected canes was not reduced and some canes were not wounded. The advantages and disadvantages of recording potential yield per cane are discussed.     

A technique for scoring midge blight of red raspberry, a disease complex caused by Resseliella theobaldi and associated fungi

A scoring technique is described for assessing midge blight using maps representing vascular lesions beneath periderm damaged by second generation larvae of Resseliella theobaldi . Individual canes were scored and some components of yield examined. Yield was not affected until lesions covered more than 20% of the stele surface at the base of canes. Midge blight scores increased with cane girth, probably because splitting of the cortex was most extensive in stout canes.     

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.