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.     

Effects of straddle-harvester design on cane blight (Leptosphaeria coniothyrium) of red raspberry

A new fruit catching device developed for raspberry straddle harvesters to reduce wounding of young canes and their consequent infection by Leptosphaeria coniothyrium was tested. During picking the young canes were contacted only by the foam rubber on a pair of belts which moved backwards along the harvester at the same speed that the machine advanced. The incidence of canes with cane blight lesions in 1981 was 18% following the use of the new device in 1980, and 79% following the use of a conventional commercial harvester equipped with overlapping metal fruit-catching plates. In comparison with the latter plates, the new device avoided wounding the cane surface directly: when the belt marks on these canes were inoculated with mycelium, however, the disease incidence was 77%. The new device caused wounds in 1980 by ripping leaves from nodes. In comparison with the commercial harvester the new device led to smaller lesions in 1981 and a lower percentage of canes dead above the fruit-catching zone. A modified new catching device tested in 1982 made barely detectable pressure marks on the young canes without bruising them. Only 6·6% of these canes developed vascular lesions after inoculation, an incidence similar to that for unwounded control canes (2·5%). The modified new device is advised for regions prone to cane blight where the cropping system has young and fruiting canes growing together.     

Further studies on resistance to Leptosphaeria coniothyrium in the red raspberry and related species

Rubus pileatus and its F₁ hybrids with raspberry (R. idaeus) were resistant to cane blight (Leptosphaeriu conioth-yriurn), but little resistance was obtained in subsequent backcross generations apart from a hybrid identified in the second backcross. Two hybrids from backcrossing R. coreanus to raspberry also showed resistance. R. pileatus and its F, hybrids produced hard growth, unlike that of raspberries, which may have been associated with a form of resistance that could not easily be transferred to commercial raspberry cultivars. Some of the genotypes used as parents showed intermediate levels of resistance and it is possible that the highly resistant genotype identified in the second backcross arose from a recombination of genes for resistance. Plants with pubescent canes (gene H) were up to 20% more resistant to mycelial inoculation than those with non-pubescent canes (gene h), and the percentage of machine-harvester inflicted wounds with disease symptoms that resulted from natural infection was also less in genotypes with pubescent canes. Many genotypes with intermediate levels of resistance suffered only limited damage from mycelial inoculation and so there are good prospects for breeding cultivars with an effective resistance, despite the limited value of R. pileatus as a donor species.     

Cane blight (Leptosphaeria coniothyrium) in mechanically harvested red raspberry (Rubus idaeus)

Leptosphaeria coniothyrium, the cane blight pathogen, was the fungus most commonly isolated from vascular lesions developing from mechanical harvester wounds on first-year red raspberry canes. When inoculated to scalpel wounds, it induced similar lesions which were later associated with bud failure and cane death as were infected wounds on machine damaged canes. Cane resistance increased from May until August, inoculations later than July inducing only small lesions and rarely causing bud failure.     

The effect of sprays of thiophanate–methyl on cane diseases and yield in red raspberry, with particular reference to cane blight (Leptosphaeria coniothyrium)

Spur blight (Didymella applanata), cane botrytis (Botrytis cinerea) and cane blight (Leptosphaeria coniothyrium) were studied in two consecutive seasons in unsprayed raspberries and in plots sprayed with thiophanate-methyl in the first season alone, (a) twice pre-harvest, (b) twice post-harvest, or (c) twice pre- plus twice post-harvest. Pre-harvest, but not post-harvest sprays, gave moderate control of both spur blight and cane botrytis; both diseases occurred mainly on the lower halves of canes, the latter being the less common. Cane blight occurred at the base of canes where they were wounded by old cane stubs. In the first year it was severe; 37% of canes died before harvest in the unsprayed plots. The three spray programmes all decreased cane death due to cane blight and they all increased yield by c. 45% even in those plots sprayed post-harvest where spur blight was severe and not controlled. Clearly this last disease, despite its high incidence, had no effect on potential yield in this experiment. In the second year cane blight was common but less severe and the incidence of its lesions was reduced similarly by all programmes, but only 4% of canes died in control plots. Analysis of the potential yield and the sizes of lesions caused by L. coniothyrium in canes inoculated at fortnightly intervals in the previous year showed that potential yield loss occurred only when lesions girdled canes. A bimodal distribution in the lesion sizes, measured by length or girdling, indicated some endogenous control of lesion development which might explain the marked differences in the effect of cane blight on potential yield in two seasons.     

Factors affecting the development of cane blight (Leptosphaeria coniothyrium) on red raspberries in Washington, Scotland and Germany

The effects of cultivar, virulence of isolates of Leptosphaeria coniothyrium, cane maturation and wound healing were examined in a series of inoculation experiments carried out over a 3–yr period in three countries in an attempt to explain why cane blight has caused serious yield losses in machine-harvested red raspberries in Europe, but not in the Pacific Northwest of America. Three isolates of L. coniothyrium from Puyallup (USA), Dundee (UK) and Dossenheim (FRG) were pathogenic on the three test cultivars Willamette, Malling Jewel and Glen Isla in all the experiments. Isolates and cultivars differed for aggressiveness and susceptibility respectively but their ranking was dependent on the test conditions and the differences were small and unlikely to explain the differences in incidence and severity of cane blight in raspberry fields in Scotland and the Pacific Northwest. Conditions at Dossenheim were most favorable for lesion development. At all sites, canes inoculated in late summer produced shorter lesions than those inoculated earlier. A delay between wounding and inoculation produced shorter lesions than simultaneous wounding and inoculation.     

Penetration of the Periderm of Red Raspberry Canes by Leptosphaeria coniothyrium

Umwounded raspberry canes were inoculated with Leptosphaeria coniothyrium. After penetration of the epidermis the fungus invaded the cortex readily. The accumulation of mycelium in this tissue lead to a partial digestion of the middle lamellae of the outermost cork layer within the polyderm. The fungus penetrated this single cell barrier through the openings between the separated cells and colonized the phelloid tissue between the first and second cork layer. After vigorous growth in this non-suberized tissue the fungus penetrated the second cork barrier in the same way as the first layer. This mode of penetration was repeated until the fungus had spread beyond the periderm and into the vascular tissues. The invasion process occurred rather slowly and was enhanced by weakening of the canes by defoliation at the time of inoculation.     

Fungi on red raspberry from lesions associated with feeding wounds of cane midge (Resseliella theobaldi)

Two types of vascular lesion are described from the base of canes in plantations infested by raspberry cane midge ( Resseliella theobaldi ); (1) brown lobate lesions ('patches') confined to midge feeding areas, (2) brown lesions spreading proximally and distally from the point of infection ('stripes'). Either or both types of lesion may be presented in individual canes. Isolations from (1) produced principally Fusarium avenaceum ; isolations from tissues where (1) and (2) are contiguous gave Leptosphaeria coniothyrium and F. avenaceum ; isolations from (2) arising from old cane stub wounds in the absence of midge gave L. coniothyrium .
The important secondary role of fungi in the midge blight complex is confirmed, but the involvement of L. coniothyrium in the complex is unclear because it also infects stub wounds on midge-infested canes.     

Effects of Didymella applanata and Botrytis cinerea on axillary buds, lateral shoots and yield of red raspberry

The viability of axillary buds and the growth and potential yield of lateral shoots at nodes of red raspberry (Rubus idaeus) infected naturally by Didymella applanata or Botrytis cinerea were measured on excised nodes, decapitated nursery canes or on canes from fruiting plantations. In comparison with lesion-free nodes, buds at infected nodes were smaller and fewer of them were capable of growth when excised and ‘forced’, although the difference in growth between infected and uninfected nodes decreased during late winter. After February, those buds at infected nodes which were capable of forced growth did so as early and with a similar growth rate as those at lesion-free nodes. In April, 70% of buds at infected nodes were capable of growth compared with 94% of those at lesion-free nodes. When nursery canes of cv. Mailing Delight were decapitated above infected nodes the emergence of lateral shoots from the terminal infected node did not differ significantly from that at lesion-free nodes. On a range of farm sites in Scotland the emergence of shoots at infected nodes in the cropping region of canes was significantly poorer than from uninfected nodes but substantially better at infected nodes of cv. Glen Clova than at those of cvs Mailing Jewel and Mailing Orion. It is suggested that cv. Glen Clova is relatively tolerant of spur blight and cane botrytis. The length and potential yield of laterals which developed at infected nodes in the cropping region of canes in these three cultivars did not differ significantly from those at lesion-free nodes. In all tests there was no significant difference in growth at nodes infected by D. applanata and B. cinerea which may indicate a common mechanism for suppression of buds.     

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.     

Effect of Didymella applanata (Niessl/Sacc.) on proline and hydroxyproline containing cell wall proteins of red raspberry

Structural cell wall proteins and their immobilisation through formation of covalent cross-links belong to important defense response mechanisms. In this work, the D. applanata — induced increase of wall-bound proline and hydroxyproline contents were associated with red raspberry resistance. The effect did not depend on the developmental stage and lignification degree of infected primocanes. Pathogen-induced accumulation of hydroxyproline noted in susceptible plants suggested the post-translational hydroxylation of proline-rich proteins and/or may be of other wall proteins. Moreover, the developmentally related increase of hydroxyproline content was associated with the resistance acquirement noted at the end of the summer period. The increase of cell wall structural protein levels was accompanied by free proline accumulation in the cytosol fraction.     

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.     

A study of ethylene in apple, red raspberry, and cherry

High ethylene levels were associated with flower abscission in apple (Malus sylvestris) and cherry (Prunus avium and Prunus cerasus), “June drop” of immature cherries, and harvest drop of apple and red raspberry (Rubus idaeus). However, an increase in ethylene content was not associated with June drop of apples and harvest drop of cherries. During the period of fruit ripening on the plant, the largest increases in ethylene occurred in apple flesh and red raspberry receptacular tissue. Ethylene remained low throughout the period of sweet and tart cherry ripening. The data obtained indicated marked ethylene gradients between adjacent tissues. Increases of ethylene in some tissues may have resulted from ethylene diffusion from adjacent tissues containing high levels of ethylene.     

Further studies on breeding for resistance to Botrytis cinerea in red raspberry canes

A high level of resistance to cane botrytis caused by Botrytis cinerea was transferred from Rubus pileatus to the red raspberry through three generations of backcrossing. The strength of the resistance showed little diminution through these generations and it was therefore thought that a major gene for resistance had been transferred. But discontinuity in the resistance levels observed was detected in plants of only one group of the progenies and so evidence for a major gene is lacking. The results showed the considerable influence of gene H, which confers pubescence, and emphasised that resistance must be separately assessed for pubescent and non-pubescent segregates.     

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)     

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.     

Resistance to raspberry cane midge (Resseliella theobaldi) and its association with wound periderm in Rubus crataegifolius and its red raspberry derivatives

Rubus parviflorus, R. odoratus and F₂ plants from the cross R. crataegifolius x R. idaeus were resistant to the raspberry cane midge (Resseliella theobaldi) when exposed to the pest in an insectary or in the field. Histological studies of R. crataegifolius and one of its hybrids with R. idaeus showed that resistance was associated with a wound periderm of suberised and lignified cells. These cells were formed from the primary cortex and phelloid cells of the polyderm as a rapid response to the occurrence of natural splits and wounds which are the oviposition sites of R. theobaldi. In the hybrid, new tissues from this wound periderm prevented the peeling of the mature primary cortex, thereby precluding serious midge attack, and resulted in a reticulate appearance on the cane surface, a distinctive cane feature useful for identifying resistant segregates.     

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.