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Red raspberry genotypes (Rubus idaeus L.) were evaluated for resistance to root rot at two field sites in Washington state and in a greenhouse study. Thirteen raspberry genotypes were planted in two field sites naturally infested with Phytophthora fragariae var. rubi Wilcox and Duncan and evaluated over 3 years for growth and symptom expression. In greenhouse pot tests, 14 genotypes were inoculated with an isolate of P. fragariae var. rubi at three inoculum levels and evaluated for growth, root color, and symptom expression using a 1 to 4 rating scale. Eleven of the 14 cultivars were found to be susceptible or very susceptible to root rot in the field and greenhouse. ‘Summit’ and ‘Newburgh’ possessed high levels of resistance to the pathogen. ‘Cascade Bounty’ also showed high resistance to root rot in the greenhouse, but confirmation from a field study is needed. Subjective root ratings of greenhouse-grown plants correlated well with measurements of cane numbers and cane infection in the field. The greenhouse tests were useful in identifying resistant genotypes and very susceptible genotypes but did not always match field results. Observation of at least 3 years in the field was necessary to compare relative reaction with root rot among genotypes.

Isozyme analysis and cytochrome oxidase sequences were used to examine whether differentiation of P. fragariae var. fragariae and P. fragariae var. rubi at the variety level is justified. In isozyme studies six strains of both P. fragariae varieties were analyzed with malate dehydrogenase (MDH), glucose phosphate isomerase (GPI), aconitase (ACO), isocitrate dehydrogenase (IDH) and phosphogluconate dehydrogenase (PGD), comprising altogether seven putative loci. Five unique alleles (Mdh-1(A), Mdh-2(B), Gpi(A), Aco(B) and Idh-1(B)) were found in strains of P. fragariae var. fragariae, whereas five unique alleles (Mdh-1(B), Mdh-2(A), Gpi(B), Aco(A) and Idh-1(A)) were present in strains of P. fragariae var. rubi. It was inferred from these data that there is no gene flow between the two P. fragariae varieties. Cytochrome oxidase I (Cox I) sequences showed consistent differences at 15 positions between strains of Fragaria and Rubus respectively. Based on isozyme data, cytochrome oxidase I sequences, and previously published differences in restyriction enzyme patterns of mitochondrial DNA, sequences of nuclear and mitochondrial genes, AFLP patterns and pathogenicity, it was concluded that both specific pathogenic varieties of P. fragariae are reproductively isolated and constitute a distinct species. Consequently strains isolated from Rubus idaeus are assigned to Phytophthora rubi comb. nov.

In winter 2002/2003, a total of 136 root samples from 57 different raspberry stocks in Scotland were examined for the presence of raspberry root rot caused by the fungus-like pathogen Phytophthora fragariae var. rubi. All stocks had been planted as propagation material entered at different grades in the Scottish certification scheme or had applied for plant passports. For detection, a modified ‘Duncan bait test’ was compared to a nested PCR method. The two tests identified the same infected stocks: PCR detected 10 positive samples from four different stocks, while the bait test picked up two additional positive samples coming from the same four stocks. The two tests had a similar level of reliability in this examination and a recommendation for one or the other depends mainly on the technical equipment and skills available in the laboratory.

A hydroponic method was developed and tested for screening red raspberry geno- types for resistance to Phytophthora fragariae var. rubi, the most common causal agent of Phytophthora root rot in raspberry. Plants of 'Titan' and 'Encore' exhibited typical disease symptoms, with the latter developing signifi cantly smaller stem lesions and fewer petiole lesions. The resistant cultivar, 'Latham', regenerated healthy root tissue from the crown and older-order roots after initial infection and necrosis of young roots and exhibited no other symptoms beyond minor leaf chlorosis. This component of the resistance reaction has not been documented previously. A segregating F 1 population from the cross of 'Latham' × 'Titan' had a survival rate of 56% with 42% classifi ed as resistant, exhibited minimal symptoms, and produced varying amounts of healthy root tissue. This screening method allows multiple observations of all plant tissues, including roots, under repeatable and defi nable growth chamber conditions. It should be useful for classifying the phenotype of individuals in segregating red raspberry populations to investigate the inheritance of Phytophthora root rot resistance using molecular markers. Phytophthora root rot is a severe disease of red raspberry (Rubus idaeus L.) in nearly all

Root rot caused by Phytophthora fragariae var. fragariae and P. fragariae var. rubi are major concerns in strawberry and raspberry production in the Pacific Northwest. Of lesser importance is black root rot of strawberry, caused by a complex of fungi and nematodes. Soil solarization was evaluated in 1997 in a strawberry planting and in 1998 in a raspberry planting for: (i) enhancing plant health and growth, and (ii) reducing population densities of root-destroying pathogens. Plots were solarized from mid-July to mid-September. Maximum and mean soil temperatures in solarized plots recorded at 10 cm depth were 48 and 33°C in the strawberry plots and 46 and 29°C in the raspberry plots. These temperatures were 7 to 17°C higher than temperatures recorded in nonsolarized plots. Soil collected after solarization was assayed by growing bait plants, cv. Totem strawberry or cv. Qualicum raspberry, at 15°C for 6 weeks in saturated soil to promote infections. Root health and plant growth were evaluated after 6 weeks. Solarization significantly reduced (P < 0.05) root necrosis and increased root weight of bait plants compared to plants grown in soil from nonsolarized plots. Infection of strawberry roots by P. fragariae, Pythium, Rhizoctonia, and Cylindrocarpon spp. was reduced (P < 0.05) by solarization in sampled soil. Disease was reduced in cv. Hood strawberries and Qualicum and Skeena red raspberries planted in solarized field plots. In the second growing season, total number and number of healthy primocanes of Qualicum plants were greater (P < 0.05) in solarized plots compared to nonsolarized plots. Solarization combined with applications of mefenoxam was no more effective in controlling diseases than solarization alone, but better than mefenoxam alone. Skeena plants responded similarly, but the differences were not significant. Red raspberry plants growing in solarized soil yielded more fruit than plants growing in nonsolarized soil in the third year after solarization. Solarization has potential as a component in an integrated pest management program of root diseases in raspberry and strawberry production, particularly within the first 2 years following planting.

A commercial serological, multiwell assay kit was used to assess the detection limits of Phytophthora fragariae var. rubi in raspberry roots. Detection limits in time lapse after inoculation, were assessed after inoculation of root systems of raspberry plants by zoospores of P. fragariae var. rubi. In extracts taken 3-9 days after inoculation, the pathogen was detected from the fourth day after inoculation. In a test series of simulated P. fragariae var. rubi infection where 0.25, 0.5, 1.0 and 1.5% of infected root tissue respectively, were mixed with healthy tissue (w/w), it was possible to detect the pathogen at 0.25% of simulated infection level. The results obtained show the possibility of an early detection of small amounts of antigen by the ELISA test procedure used. This enhance possibilities for early diagnosis and thereby more effective prevention of Phytophthora diseases in raspberry.

The internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal RNA gene repeat from Phytophthora species were amplified using the polymerase chain reaction and sequenced. Sequences from P. cambivora, P. cinnamomi, P. citricola, P. cryptogea, P. drechsleri, P. fragariae var. fragariae, P. fragariae var. rubi, P. megasperma var. megasperma and P. nicotianae were compared with published sequences and phylogenetic trees were produced. The resultant grouping of species generally agreed with groupings established using classical morphological criteria, primarily sporangial morphology. Amongst species with non-papillate sporangia two clades were evident, one consisting of P. fragariae, P. cambivora and P. cinnamomi and the other of P. megasperma, P. drechsleri and P. cryptogea. The latter three were placed in the tree between the non-papillate groups and the papillate and semi-papillate groups which formed three distinct clades. One group comprised P. citricola, P. citrophthora and P. capsici, another P. megakarya and P. palmivora and a third P. pseudotsugae, P. cactorum, P. idaei, P. nicotianae and P. infestans. More isolates of P. megasperma, P. drechsleri and P. cryptogea will need to be examined to settle more precisely the relationship of these species to the others. PCR amplification of ITS sequences using freeze-thawed mycelial scrapings from pure cultures growing on agar followed by digestion with restriction enzymes is a quick and easy way to compare and identify isolates without the need for laborious DNA extraction procedures. With improved technology, rapid automatic sequencing of PCR-amplified ITS regions is now possible and yields detailed information of relationships within the genus as well as allowing the design of species-specific PCR primers for diagnostic purposes.

A collection of about 200 actinomycete strains was screened for the ability to grow on fragmented Phytophthora mycelium and to produce metabolites that inhibit Phytophthora growth. Thirteen strains were selected, and all produced (beta)-1,3-, (beta)-1,4-, and (beta)-1,6-glucanases. These enzymes could hydrolyze glucans from Phytophthora cell walls and cause lysis of Phytophthora cells. These enzymes also degraded other glucan substrates, such as cellulose, laminarin, pustulan, and yeast cell walls. Eleven strains significantly reduced the root rot index when inoculated on raspberry plantlets.

The inheritance of root rot resistance in red raspberry, caused by Phytophtora fragariae var. rubi, was investigated using two experimental breeding designs. Design A was not randomly mated and genetic parameters could not be calculated. Design B was therefore randomly mated to make it possible to do such estimations. Considerable genetic variation was associated with the recorded traits on seedlings tested in a glasshouse. Disease index and % survival were both found to be highly heritable with broad sense heritabilities (H2) close to 0.9 in each design. Estimates of narrow sense heritability (h2), indicated that progress towards resistance would be moderate. In the first design the cultivars ‘Festival’, ‘Asker’, ‘Boyne’ and ‘Sentry’ and the clones N85-02-13 and N85-06-140 had high general combining abilities. Some crosses had a high specific combining ability indicating the presence of non-additive genetic variation.

Severe root rot of raspberry is caused in Europe and North America by a homothallic, non-papillate Phytophthora sp., which has been identified by different investigators as P. erythroseptica, P. fragariae or as a highly pathogenic variant of P. megasperma . Two collections of such highly pathogenic raspberry isolates from Europe and North America were compared with recognized specimens of the three above named species, which were originally isolated from potato, loganberry and strawberry, and a variety of perennial host plants, respectively. All such raspberry isolates, regardless of previous identification, formed an essentially homogeneous group with respect to colony and growth characteristics, the production and morphology of gametangia, the morphology and dimensions of sporangia, electrophoretic banding patterns of mycelial proteins and pathogenicity. They were readily distinguishable from recognized isolates of P. erythroseptica and P. megasperma with respect to cultural, morphological, and electrophoretic criteria. In contrast, they were very similar to isolates of P. fragariae from strawberry (and a single isolate from loganberry) although the two groups could be separated by differences in growth rate on some agar media, production of oospores in culture, and small differences in electrophoretic banding patterns, as well as in pathogenicity. It was concluded that the raspberry isolates should be assigned to P. fragariae , but that they should be given a subspecific epithet at the varietal level to distinguish them from strawberry isolates of the species i.e. P. fragariae var. rubi .

The following chapter describes a PCR method for the identification of the raspberry root rot pathogen Phytophthora fragariae var. rubi. Furthermore, a nested PCR suitable for the detection of the pathogen in infected raspberry roots and validated against the "Duncan bait test" (EPPO Bull 35:87-91, 2005) is explained. Protocols for different DNA extraction methods are given which can be transferred to other fungal pathogens.

Titan' red raspberry (Rubis idaeus L.), highly susceptible to root rot caused by Phytophthora fragariae Hickman var. rubi Wilcox & Duncan (syns. P. erythroseptica Pethyb., "highly pathogenic" P. megasperma Drechs.), was planted in June 1990 in a silt loam naturally infested with the pathogen. Raked beds (0.36 m high) dramatically reduced disease incidence and severity relative to flatbed treatments. In contrast, metalaxyl at 372 mg·m -1 of row provided little benefit when applied to flat beds and provided consistently moderate but statistically insignificant effects when applied to raised beds. Relative to the flat bed system, primocane vigor was increased in 1992 by 16%, 190%, and 224% in the flat bed plus metalaxyl, raised bed, and raised bed plus metalaxyl treatments, respectively; total yields were increased by 7%, 231%, and 272% with these same respective treatment. The results indicate that raised-bed planting systems can provide substantial control of phytophthora root rot of red raspberries even when highly susceptible varieties are grown on otherwise marginal sites. Metalaxyl appears more effective as a supplement rather than substitute for raised beds under such conditions. Chemical name used: N- (2,6- dimethylphenyl) -N- (methoxyacetyl)alanine methyl ester (metalaxyl). During the 1980s, phytophthora root rot became recognized as a major cause of declin- ing red raspberry plantings in commercial production areas worldwide. A general pro- gram has been proposed for controlling the disease (Wilcox, 1991)—one that integrates pathogen exclusion, site modifications to pro- mote water drainage (e.g., raised-bed planting systems), use of partially resistant cultivars, and treatment with specific fungicides. How- ever, this program's individual components are not equally practical or desirable. For instance, many raspberry fields are now con- taminated with pathogenic Phytophthora spp., precluding the possibility of pathogen exclu- sion when such sites are replanted. Further- more, some cultivars, which are otherwise among the most profitable and horticulturally desirable, are also highly susceptible to the disease (Barritt et al., 1981; Wilcox and Nevill, 1992). Therefore, the purpose of this experi- ment was to examine the efficacies of a raised- bed planting system and registered fungicide treatment, both singly and in combination, when replanting a highly susceptible cultivar in a pathogen-infested site. The experiment site, located near Geneva, N.Y., on Lima silt loam soil, was nearly level. A previous raspberry planting was removed from the site in 1989, following a prolonged

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623–628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

Onze souches d'actinomycetes ayant la capacité de protéger les plants de framboisiers (Rubus strigosus) contre les infections causées par les Phytophthora ont été caractérisées. Il a été montré que toutes les souches appartenaient au genre Streptomyces. Deux souches (EF-34 et EF-76) croissaient à 4, 15 et 30°C sur un milieu V8 agar dont le pH avait été ajusté entre 5 et 9. Sept souches dont EF-34 et EF-76 pouvaient hydrolyser les parois cellulaires de Phytophthora et inhiber la croissance du champignon à 15°C et à des pH variant entre 5 et 9. Toutes les souches inhibaient la croissance du P. fragariae var. rubi et du Pythium ultimum. La croissance d'autres espèces fongiques et de bactéries à Gram négatif n'était inhibée qu'en présence de trois souches (EF-14, EF-72 et EF-76). Les onze actinomycetes antagonistes ont été classés en quatre groupes selon leur résistance à divers pesticides utilisés pour protéger les cultures de framboisiers. La souche EF-76 a été caractérisée plus en détail. Cette souche a été identifiée comme étant le Streptomyces hygroscopicus var. geldanus, et produisait l'antibiotique geldanamycine., Eleven actinomycete strains that were previously shown to protect raspberry (Rubus strigosus) plants against Phytophthora infection were characterized. all were shown to belong to the genus Streptomyces. Two strains (EF-34 and EF-76) grew at 4, 15 and 30°C on V8 agar between pHs 5 to 9. Seven strains including EF-34 and EF-76 had both the ability to hydrolyze Phytophthora cell walls and to inhibit Phytophthora growth at 15°C between pHs 5 to 9. all actinomycetes inhibited the growth of P. fragariaevar. rubi and of Pythium ultimum. The growth of other fungal species and of Gram-negative bacteria was inhibited only in the presence of three strains (EF-14, EF-72, and EF-76). The eleven antagonistic actinomycetes were classified into four groups with regard to their resistance to various pesticides used to protect raspberry crops. Strain EF-76 was further characterized. This strain was identified as Streptomyceshygroscopicus var. geldanus, and it was shown to produce geldanamycin, a known antibiotic.