A gene-for-gene model to explain interactions between cultivars of strawberry and races of Phytophthora fragariae var. fragariae

 A gene-for-gene model is postulated to explain the observed interactions between cultivars of strawberry and races of Phytophthora fragariae. Five interacting resistance (R1–R5) and avirulence (Avr1–Avr5) factors explain all the available data involving 15 host genotypes, including the USA and Canadian differential series, and 12 pathogen isolates from North America. Interactions between pathogen isolates and UK and German differentials are also explained by the proposed model. The model makes it possible to develop a universally applicable differential series, to present a systematic, unequivocal nomenclature of races, and to increase the efficiency of breeding programs. Received: 26 April 1996 / Accepted: 19 July 1996     

Resistance to Phytophthora fragariae var. fragariae in strawberry: the Rpf2 gene

  Phytophthora fragariae var. fragariae is the causal agent of red stele (red core) root rot in strawberry (Fragaria spp.). The inheritance of resistance to one isolate of this fungus was studied in 12 segregating populations of F.×ananassa derived from crosses between four resistant cultivars (‘Climax’, ‘Redgauntlet’, ‘Siletz’, and ‘Sparkle’) and three susceptible cultivars (‘Blakemore’, ‘Glasa’, and ‘Senga’ Sengana’). The analysis clearly supports the hypothesis of a single segregating dominant resistance gene. It is proposed that this gene be designated Rpf2. Received 12 November 1996 / Accepted: 22 November 1996     

Analysis of Cross-Reactive Antigens of Phytophthora fragariae and Strawberry and their Relation to Resistance and Susceptibility

The presence of cross-reactive antigens between five isolates of P, fragariae (Pf 1, Pf 2, Pf 3, Pf 10 and Pf 11) belonging to five physiological races of the fungus and five strawberry cultivars (Cambridge Favourite, Hapil, Red Gauntlet, Siletz and 52AC18) exhibiting different disease responses to the five isolates was demonstrated by Western blotting. Antiserum anti-H, raised against extracts of healthy Cambridge Favourite roots, detected two antigens which were common to all isolates. Concentration of one of these antigens might be related to the pathogenicity of P. fragariae isolates. Antiserum anti-PfM, raised against mycelial suspensions from the five isolates, detected a doublet of 64 and 61 kDa in the soluble extracts of healthy roots from the five cultivars tested. The corresponding root suspensions revealed numerous other antigens which reacted with anti-PfM. In susceptible interactions, the doublet described above and two additional polypeptides of 38 and 31 kda were detected in large concentrations in both the soluble extracts and root suspensions. These four polypeptides were shown to be present in the healthy roots as well as in the mycelial extracts. In resistant interactions, a 116 kDa polypeptide, present in the root suspensions of the healthy host, was detected in the soluble extracts of the infected roots. It is suggested that these antigens might have a role in resistance and susceptibility.     

Sensitivity of the ELISA Test to Detect Phytophthora fragariae var. rubi in Raspberry Roots

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.     

Identification of RAPD markers linked to a Phytophthora fragariae resistance gene (Rpf1) in the cultivated strawberry

 Bulked segregant analysis (BSA) was used to identify seven random amplified polymorphic DNA (RAPD) markers linked to the Rpf 1 gene. Rpf 1 confers resistance to Phytophthora fragariae var. fragariae, the causal agent of red stele root rot in Fragaria spp. The bulked DNAs represented subsets of a F₁ population obtained from the cross Md683×Senga Sengana which consisted of 60 plants and segregated in a 1:1 ratio for resistance or susceptibility to race 2.3.4 isolate NS2 of P.  fragariae. Seven markers were shown to be linked to Rpf 1 and were generated from four primers; five of these markers were in coupling phase and two in repulsion phase with respect to the gene. A linkage map of this resistance gene region was generated using JoinMap 2.0^TM. The manner in which Rpf 1 and the linked markers co-segregated indicated that they are inherited in a disomic fashion. These markers could enable gene pyramiding and marker-assisted selection of resistance genes in strawberry breeding programmes. Received: 26 August 1996 / Accepted: 20 December 1996     

Discrimination between viable and dead Xanthomonas fragariae in strawberry using viability PCR

Xanthomonas fragariae is the causal agent of an important bacterial disease in strawberry production regions worldwide and a quarantine plant pathogen in many countries including New Zealand. Xanthomonas fragariae mainly infects the foliage of strawberry plants but can also infect the calyx tissue associated with strawberry fruit. Fresh strawberries are a high-value internationally traded commodity that has a short shelf-life. When making biosecurity decisions based on the finding of a quarantine organism such as X. fragariae by PCR, one of the major challenges is the inability to differentiate positive results originating from viable or dead cells. Viability PCR (vPCR) is a technique that selectively inhibits PCR amplification of DNA derived from dead cells through the use of a nucleic acid intercalating dye, for example, PEMAX™. A vPCR protocol has been optimized to enable rapid detection of viable X. fragariae in a tissue sample. PEMAX™ treatment resulted in complete inhibition of PCR amplification of 10⁸–10³ cfu/ml dead X. fragariae cells in strawberry host tissue. The most important parameters for optimization were the dilution of the sample, amplicon length and choice of nucleic acid intercalating dye. This study provides a rapid protocol to discriminate between viable and dead X. fragariae in strawberry in a phytosanitary environment. This test will help timely decisions to be made at the border on imported fresh strawberry consignments that test positive for X. fragariae.     

Nachweis von Phytophthora fragariae Hickman in Wurzeln der Erdbeerkultursorte 'Tenira' mit Hilfe des enzyme-linked immunosorbent assay (ELISA)

Detection of Phytophthora fragariae Hickman in roots of strawberry cultivar ‘Tenira’ by enzyme-linked immunosorbent assay (ELISA) Phytophthora fragariae Hickman is detected by ELISA in roots of strawberry cv. ‘Tenira’. Because of the high sensitivity of ELISA presence on fungal antigen was demonstrated before symptoms are detected in microscopical investigations.     

Growth and reproduction in culture of ten Phytophthora fragariae races

Cultural characteristics were employed to develop a basis for characterizing each of the ten recognized races ofPhytophthora fragariae Hickman in the United States. Investigations were made into the relationships of temperature to growth and colony morphology, the effects of various media and ß-sitosterol on growth and oospore production, and relative zoosporangium production capabilities of the ten races. On the basis of this information, the ten races were divided into two groups according to their colony morphologies. Further characterization was possible on the basis of differential oospore and zoosporangium production in different media, and, to a lesser extent, with contrasting increments in mycelial mass and linear extension.     

Genotype-dependence in the interaction betweenGlomus fistulosum,Phytophthora fragariae and the wild strawberry (Fragaria vesca)

Root colonisation byGlomus fistulosum BEG 31 of a plant population from the outbreeding wild strawberry (Fragaria vesca) ranged from 42 to 80%; this was correlated with a significant increase in fruit trusses, berries and leaves. The mycorrhizal plants produced significantly more primary and secondary runners but less lateral runners. One hundred randomly selected seeds were multiplied in vitro and 4 clonal populations were selected. Root colonisation in the clones varied from 16 to 75%, with significant difference in the % colonisation between some of the clones. Percentage mycorrhizal colonisation was positively correlated with effects on host growth.The positive correlation between mycorrhizal root colonisation and growth effects in the clones was not reflected in their respective susceptibility, in the non-mycorrhizal state, toPhytophthora fragariae infection. Clones showing the highest and lowest mycorrhizal root colonisation showed high disease susceptibility whereas a clone with intermediate colonisation was resistant. When the clones were colonised with mycorrhizal fungi, the two susceptible clones became resistant toP. fragariae whereas the resistant and partially-resistant clones were less affected. Only one clone showed variation in vesicle formation when challenged withP. fragariae.     

Interaction of Pratylenchus penetrans and Rhizoctonia fragariae in Strawberry Black Root Rot

A split-root technique was used to examine the interaction between Pratylenchus penetrans and the cortical root-rotting pathogen Rhizoctonia fragariae in strawberry black root rot. Plants inoculated with both pathogens on the same half of a split-root crown had greater levels of root rot than plants inoculated separately or with either pathogen alone. Isolation of R. fragariae from field-grown roots differed with root type and time of sampling. Fungal infection of structural roots was low until fruiting, whereas perennial root colonization was high. Isolation of R. fragariae from feeder roots was variable, but was greater from feeder roots on perennial than from structural roots. Isolation of the fungus was greater from structural roots with nematode lesions than from non-symptomatic roots. Rhizoctonia fragariae was a common resident on the sloughed cortex of healthy perennial roots. From this source, the fungus may infect additional roots. The direct effects of lesion nematode feeding and movement are cortical cell damage and death. Indirect effects include discoloration of the endodermis and early polyderm formation. Perhaps weakened or dying cells caused directly or indirectly by P. penetrans are more susceptible to R. fragariae, leading to increased disease.     

Genetic Diversity of Xanthomonas arboricola pv. fragariae Strains and Comparison with some other X. arboricola Pathovars using Repetitive PCR Genomic Fingerprinting

The genetic relationship within 26 Xanthomonas arboricola pv. fragariae strains and between this pathovar and 20 strains of X. arboricola pv. corylina, 22 strains of X. arboricola pv. juglandis and 16 strains of X. arboricola pv. pruni has been assessed by means of repetitive polymerase chain reaction (rep‐PCR) using Enterobacterial Repetitive Intergenic Consensus), BOX (BOXA subunit of the BOX element of Streptococcus pneumoniae) and repetitive extragenic palindromic primer sets. Cluster analysis was performed by means of unweighted paired group method using arithmetic average (UPGMA). Upon rep‐PCR and UPGMA cluster analysis, a relevant genetic diversity was found within the strains. The overall similarity, however, was high (i.e. 80%). The four X. arboricola pathovars showed similar but clearly different genomic patterns and clustered into four different groups, with X. arboricola pv. corylina and X. arboricola pv. juglandis more closely related to X. arboricola pv. fragariae. Representative strains of X. arboricola pv. fragariae and the putative xanthomonads isolated from strawberry leaves showing leaf blight symptoms underwent pathogenicity tests. After artificial inoculation, X. arboricola pv. fragariae induced necrotic spots accompanied, sometimes, by a chlorotic halo. The blackening of the leaf veins and peduncle was, sometimes, also observed. The four putative xanthomonads isolated from diseased strawberry leaves and not inducing symptoms after artificial inoculation, clustered apart from X. arboricola pathovars.     

Ruscus hypophyllum: A New Host for Aphelenchoides fragariae

Aphelenchoides fragariae was isolated from the phylloclades of the ornamental plant Ruscus hypophyllum (Liliaceae). Rotylenchus buxophilus, Scutellonema brachyurum, and Meloidogyne were identified as the most common plant-parasitic nematodes in the soil near the roots. The pathology and life history of A. fragariae were closely related to the climate. To our knowledge, this is the first report of R. hypophyllum as a host of plant-parasitic nematodes.     

The effect of Tarsonemus fragariae Zimmermann (Acarina: Tarsonemidae) on strawberry yields

Experimental plots, arranged within commercial strawberry fields, were used to study the effect of Tarsonemus fragariae infestations on strawberry yields. Yields and the mean weight of individual fruits were inversely related to mite infestation levels. Yield reductions of over 50% were attributable to this pest.     

Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae

Background and aims

Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars.

Methods

Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4?h to 7?d post inoculation (pi).

Results

Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues.

Conclusions

This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder.     

Pathotypes of Peronospora viciae in Britain

Fifty-five isolates of Peronospora viciae from a wide range of locations in Britain were tested on 10 inbred lines of Pisum sativum. Reactions ranged from extreme resistance, where the pathogen failed to spread in the tissues and caused no macroscopic symptoms, to extreme susceptibility, where the pathogen spread extensively and sporulated abundantly. Based on two differential reactions, namely, symptomless resistance and all other more susceptible reactions, 22 groups of isolates were distinguishable. On the four most discriminating differential lines, eleven groups of isolates were distinguished; these are designated UK Pathotypes, (UKP) 1–11, and the four lines nominated as standard differential hosts.     

Evidence for Differential Resistance to Peronospora parasitica (downy mildew) in Accessions of Brassica juncea (mustard) at the Cotyledon Stage

Thirty-one Brassica juncea accessions were screened at the cotyledon stage for resistance to four isolates of Peronospora parasitica. Isolates R1 and P003 were derived from crops of oilseed rape (B. napus ssp. oleifera) in the UK and isolates IP01 and IP02 were derived from crops of mustard (B. juncea) in India. B. napus cv. Ariana, which was used as a susceptible control for isolates from B. napus, was resistant to isolates from B. juncea. All, B. juncea accessions were resistant to isolates from B. napus except one accession which expressed moderate resistance to isolate P003. Five groups of B. juncea accessions with differential resistance were identified. Lines homogeneous for resistance were selected from seedling populations of accessions that exhibited a heterogeneous reaction to isolates from B. juncea. The differential resistance identified in the B. juncea-P. parasitica combination can be used as a foundation for future studies of the genetics of the host-pathogen interaction and for breeding for disease resistance.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

Pectic enzymes as a selective pressure tool forin vitro recovery of strawberry plants with fungal disease resistance

Summary A novel strategy in selecting strawberry (Fragaria xananassa L.) plants with resistance toRhizoctonia fragariae andBotrytis cinerea was developed. Purified pectic enzymes produced byR. fragariae were usedin vitro to select morphogenetic calluses. Both regenerated shoots and plants were testedin vitro andin vivo withR. fragariae andB. cinerea. Thein vitro resistance of shoots regenerated under selection pressure was confirmed byin vivo tests with runner plants either by root immersion in a suspension ofR. fragariae mycelium before potting the plants in sterile soil, or by spraying the leaves with several strains ofB. cinerea spores. The increase of resistance against pathogens was correlated to the increase of phenolic compounds, particularly orthodibydroxyphenolsAbbreviations MS (Murashige & Skoog 1962) - BAP (6-benzylaminopurine) - IBA (Indole-3-butyric acid) - PE (Pectic enzymes) - PDA (Potato dexstrose agar) - PG (Polygacturonase) - RU (Reducing Units) - SE (Standard Error) Communicated by N. Amrhein     

Genetic control of immunity to turnip mosaic virus in winter oilseed rape (Brassica napus ssp. oleifera) and the effect of foreign isolates of the virus

Immunity to a UK isolate (UK 1) of turnip mosaic virus (TuMV) was studied in eight lines of oilseed rape selected from cv Rafal. Six of these lines were uniformly immune and two segregated. Segregation ratios in the F₂ generation of reciprocal crosses between two uniformly immune lines and two uniformly susceptible cultivars (Mikado and Yeoman) showed that immunity was controlled by a dominant nuclear allele. The immunity was confirmed by the inability to detect virus particles in mechanically inoculated plants by back inoculations, ELISA and ISEM tests. Plants were immune to repeated inoculations and aphid transmissions. The immunity was effective against one other UK isolate and two German isolates of TuMV. Another UK isolate (UK 3) and a Greek isolate partially overcame the immunity causing local infection and a Canadian and a Danish isolate overcame it completely causing systemic mosaic-type symptoms. When these immunity-breaking strains were tested against swede line 165 which is also immune to UK 1 TuMV, the Canadian isolate overcame the immunity whereas the Danish isolate did not. Using this swede line, one susceptible and one immune line of oilseed rape as differentials, four distinct groups of TuMV isolates could be identified. Selections of oilseed rape immune to UK TuMV isolates were more severely affected by the Canadian TuMV than UK TuMV susceptible selections. The gene determining immunity to TuMV had no pleiotropic effect on susceptibility to cauliflower mosaic virus. The implications on these findings in relation to breeding for virus resistance are discussed.