P. H. Gregory Paper Reading Competition Abstracts
Development of host resistance to Phytophthora pod rot disease of
cocoa, is there hope for the future?
The economies of 57 tropical countries world-wide particularly in West and Central Africa largely depends on cocoa (Theobroma cacao, L) production, the raw material for chocolate manufacture. Unfortunately, the sustainability of these economies and the livelihood of the majority of their small-scale farmers have been increasingly threatened by numerous factors including fungal diseases; the most important one being Phytophthora pod rot. For several decades efforts have been made to develop resistant varieties to this disease, but until recently, progress had been hampered by a number of factors. These include the lack of reliable early screening methods, variation in pathogen populations from country to country and the lack of global or regional collaborative projects to tackle these problems in an integrated manner. However, recently, significant progress has been made in overcoming these problems. This includes the development of the leaf disc inoculation technique for rapid screening of germplasm, the establishment of strong correlation between field resistance and rapid screening techniques, and the initiation of several global projects investigating genotype-isolate interaction.
This current study focused on the implications of pathogen variability and others factors such as inoculum density and the period of assessment after inoculation, on screening cocoa germplasm for resistance, using the leaf disc inoculation technique. An inoculum concentration of 3.0 x 105 zoospore/ml with assessment of lesions 7 days after inoculation was the most effective combination for the separation of clones tested.
Seven clones belonging to the Amazon Forestario parentage, MA12, GU225P, GU144C and VENC4/4 (Lower Amazon Forestario), and PA120, LCTEEN162/10 and SCA6 (Upper Amazon Forestario) showed good levels of resistance to Phytophthora pod rot. Two Trinitario hybrids (ICS48 and ICS1) and a Forestario clone, PLAYA ALTA2 were highly susceptible. Thus, cocoa materials identified from very susceptible to resistant can now be used for detailed studies of resistance mechanisms; some clones exhibited hypersensitive response to the pathogen.
The implications of these positive findings, effects of variability and distribution of the pathogen species on in-country screening programmes and the advantages of screening cocoa germplasm outside the cocoa growing countries are discussed.
Pathogenicity and crucifer isolates of Verticillium dahliae
Verticillium dahliae and V. alboatrum are important soil-borne plant pathogens causing vascular wilts in a wide range of crops throughout the world. Although more than 300 agriculturally important plants are susceptible to these two species, the majority of isolates do not infect cruciferous plants. Most isolates of both species are short (<5.5m) spored and are haploid but some classified as V. dahliae because they produce the distinctive microsclerotial resting structures are long (>7.0m) spored and have a higher DNA content. These isolates appear to be natural hybrids which probably arose through parasexual hybridisation between V. dahliae and V. alboatrum. As a means of identifying interspecific hybridisation events the presence and identity of both major and minor ITS sequences in the rRNA genes was examined by PCR amplification and direct sequencing or SSCP. Sequence analysis of 30 isolates from diverse geographical locations and hosts revealed that at least 4 different hybridisation events have occurred. Isolates arising from different hybridisation events were also found to contain different repeat structures within the intergenic spacer region of the rRNA genes. Further work using a broader genomic approach will also be used to investigate the relationships of these isolates to haploid isolates of V. dahliae and V. alboatrum and help to elucidate the molecular mechanisms governing this novel crucifer pathogenicity.
Catching the Crooks: diagnostics and phylogenetic analysis of Spongospora
subterranea f. sp. nasturtii
The plasmodiophorid organism Spongospora subterranea f. sp. nasturtii is the causal agent of crook root disease of watercress (Rorippa nasturtium-aquaticum). The only current control measure is zinc, the use of which is restricted due to environmental concerns. Zinc is currently added to watercress beds throughout October to April in most years, and a means of rationalising applications would be beneficial to the watercress industry. Such an approach would require accurate determination of the presence and quantity of S. subterranea f. sp. nasturtii zoospores in watercress beds, but no adequate methods are currently available.
Using internal transcribed spacer (ITS) and 18S ribosomal DNA (rDNA), a PCR-based diagnostic test was developed for S. subterranea f. sp. nasturtii. Primers designed were shown to be specific, able to amplify from samples collected from a range of geographic locations, and could be used to amplify DNA directly from zoospores. Efforts to develop a sampling technique for zoospores in watercress beds were successful based on washing of root material prior to PCR.
In addition, the 18S rDNA sequence was used to infer phylogeny of S. subterranea f. sp. nasturtii. When analysed alongside other plasmodiophorids, S. subterranea f. sp. nasturtii appeared most closely related to S. subterranea f. sp. subterranea and Plasmodiophora brassicae, based on 270 bases at the 3 end of the gene. Examination of 18S rDNA sequence data from Spongospora and Plasmodiophora suggested that these form a distinct taxonomic grouping, not closely linked to either protists or fungi.
Genetic diversity among isolates of Xanthomonas hortorum pv. hederae
Bacterial leaf spot causes significant losses for commercial growers of English ivy (Hedera spp.) in the UK. Ivies represent a considerable proportion of production at many nurseries, with an ex-nursery value estimated at approximately 4 million. Seventy-one bacterial isolates were obtained from lesions on diseased ivy and five putative isolates of Xanthomonas hortorum pv. hederae (Xhh) were obtained from the NCPPB (National Collection of Plant Pathogenic Bacteria), including three isolates from ivy and two isolates from Schefflera arboricola. Fifty-four of the isolates from ivy, including the three from the NCPPB, were identified as Xanthomonas based on phenotype and were pathogenic on Hedera helix cv. Green Ripple and were therefore considered to be Xhh. Two isolates from Schefflera arboricola from the NCPPB were not identified as Xanthomonas based on phenotype and were not pathogenic on ivy or Schefflera actinophylla. The genetic diversity among 33 isolates of the pathogen Xhh, representing three countries, six different regions in the UK and sixteen different Hedera species and cultivars was examined using Random Amplified Polymorphic DNA (RAPD) PCR. Isolates of Xhh from ivy were all closely related (>76% similarity) although it was possible to distinguish three sub-groups at the 80% similarity level. However, these sub-groups did not appear to show any relationship with the geographical origin or cultivar of origin. The implications of these results for the epidemiology of this disease will be discussed.
Keywords: Bacterial leaf spot, RAPD PCR, group, cluster analysis
Omnipotent Oidium Surfaces, Signals and Sensing
Oidium lycopersici is a highly polyphagous pathogen of glasshouse grown tomatoes. The identification of this tomato powdery mildew, in the late 80s, led to a number of hypotheses as to its origin. Our recent work has revealed, from ITS sequence analysis, that O. lycopersici to have a close similarity to the Erysiphe aquilegiae var ranunculi, the buttercup powdery mildew.
Early work led to the identification of the key stages in the development of O. lycopersici. The sequence of development was followed from germination at 3 -5 hours after inoculation (h.a.i.) and differentiation between 6 - 8 h.a.i. Scanning electron microscopy has revealed specific morphological features on the conidial coat and on the appressorial body.
Host penetration has been found to result from concomitant action of force, as determined by turgor measurements by plasmolysis and cytorrhysis, and from cutinase activity, assessed using p-nitrophenyl fatty acid substrates.
An early peak in spore conidial cutinase activity was observed prior to germination, but within 1 hour of host contact. The development of a novel adhesion assay has revealed cutinase activity to be involved in early conidial adhesion. Further work has identified a second peak in cutinase activity post germination but prior to full appressorial differentiation. This peak in activity was found to be pivotal for host surface perception and subsequent differentiation, assessed by timed application of the cutinase inhibitors, the ebelactones.
Current work aims to bring this research full circle: to establish a role for surface receptors in pathogen development. RGD peptides have been used in assessment of conidial adhesion, germ tube development, and appressorial formation, in addition to the development of turgor within the appressoria.
Jones, H.E., Whipps, J.M., Thomas, B.J., Carver, T.L.W., Gurr, S.J. (2000) Initial events in the colonisation of tomatoes by Oidium lycopersici, a distinct powdery mildew fungus of Lycopersicon species. Can. J. Bot. 78: 1 - 6
Identification of genes required for N-mediated resistance against
TMV by virus-induced gene silencing
The aim of this project was to identify genes required for the N mediated defence response against tobacco mosaic virus (TMV).
Infection of plants by a virus carrying a fragment of a host gene leads to suppression of the corresponding host gene in a process termed virus induced gene silencing (VIGS). Here VIGS was exploited to identify genes required for N-mediated resistance; silencing genes necessary for N function will break resistance and enable TMV susceptibility.
Nicotiana benthamiana plants are amenable to VIGS. Thus an N genomic fragment from tobacco was used to transform N. benthamiana plants. N transgenic plants were resistant to recombinant TMV isolates demonstrating that components necessary for N function are likely to be conserved between tobacco and N. benthamiana.
In order to validate the notion that VIGS could be used as a tool to identify components of the N resistance response, N itself was targeted for suppression. Infection of N transgenic plants with virus vectors carrying a fragment of N led to silencing of N and TMV susceptibility.
The requirement of EDS1 in the N resistance pathway was then tested. VIGS of a N. benthamiana EDS1 homologue compromised N resistance; TMV replication on EDS1 silenced plants occurred to a similar extent as on N silenced plants. These observations provide evidence that EDS1 is required for function of TIR-NBS-LRR resistance genes in species other than Arabidopsis.
Finally, VIGS was used to identify a novel N resistance pathway gene. A normalised N. benthamiana cDNA library was cloned into a potato virus X (PVX) vector. 5 000 N transgenic plants were inoculated with PVX-cDNA constructs from the library to induce silencing of corresponding genes. The plants were then screened for loss of N resistance. The N response was consistently compromised by VIGS of NRG1 (for N requirement genes). NRG1 is predicted to encode a non-TIR NBS-LRR protein. Transient over-expression of NRG1 elicited a hypersensitive response in the absence of N or the elicitor of N implying that NRG1 functions downstream of N. VIGS of NRG1 in non-transgenic N. benthamiana, i.e. TMV compatible plants, did not enable enhanced TMV replication. NRG1 silencing did not suppress the resistance response mediated by Rx or by Pto.
In summary, VIGS was used to demonstrate that EDS1 is a necessary component of the N resistance response and that N function depends on another NBS-LRR encoding gene, NRG1.
Characterization of a 40kb plasmid in Pseudomonas syringae pv maculicola
involved in pathogenicity in Arabidopsis
We are interested in understanding the interactions between pathogens and their host, using as a model, Pseudomonas syringae pv maculicola strain M6 (PsmM6) on Arabidopsis. A 40kb region of PsmM6, carrying the avr gene avrRpm1, excises from the chromosome and replicates as a plasmid (FKN plasmid). Based on the sequences of the borders of the region in the chromosome and of the plasmid itself we have developed a putative mechanism for the excision and integration process. FKN has been shotgun cloned and sequenced with an average of 6-fold redundancy. The G+C content is significantly lower than in the rest of the chromosome (53.35% vs 58.5%). It harbors open reading frames with homologies to known avr genes, genes encoding transcriptional regulators, transmembrane proteins as well as proteins necessary for the maintenance of the plasmid. FKN also carries DNA sequences with homologies to mobile elements. These features are found in known pathogenicity islands in other pathogenic bacteria. The FKN plasmid has been cured from PsmM6. We are in the process of characterizing the phenotypic differences between the cured strain and PsmM6 on Arabidopsis cultivars. Preliminary results suggest that the FKN plasmid plays a role in the interaction between PsmM6 and Arabidopsis. The function of the proteins encoded by the plasmid will be discussed, as well as the potential mechanisms of integration and excision of the plasmid.
Elemental sulphur formation in plants and defence against pathogens
Elemental sulphur formation is well documented in certain specialised prokaryotes but rarely in eukaryotes. Our evidence suggests that mans oldest fungicide may function in some plants as a phytoalexin. Elemental sulphur (S0) was detected (as S8) in the xylem of resistant genotypes of Theobroma cacao and tomato to infection by the vascular pathogen Verticillium dahliae. S0 was identified and quantified (S34 standard) for the first time by GC-MS. SEM-EDX revealed accumulation of sulphur in xylem parenchyma cells and other vascular structures in potential contact with V. dahliae, which is a rare example of cellular localisation of an antimicrobial substance. Furthermore, elemental sulphur has been detected in the xylem of resistant or tolerant genotypes of tomato plants in response Ralstonia solanacearum and in tobacco and cotton plants in response to fungal vascular pathogens. S0 has not been detected in leaves of diverse plant species exhibiting the hypersensitive response to incompatible bacterial pathogens but appeared to be constitutive in leaves of Arabidopsis thaliana. Currently we are elucidating the biogenic route for S8 formation in response to infection by both biochemical and molecular techniques. Its production is by an uncharacterised pathway that may involve oxidation of sulphide. One route could be from glutathione and cysteine pools. Sulphate and thiol pools were determined by HPLC in infected, resistant tomato tissues. Glutathione increased ca. threefold in xylem and leaves during early invasion (14d) and cysteine also increased in vascular tissues at this time but only in plants grown under a high sulphur regime. Accumulation of glutathione may be linked to reduction of active oxygen species, which are rapidly generated during incompatible interactions. The effect of sulphur levels on this putative defence response may be significant in view of current sulphur defiency in many European crops. Toxicity of S8 to a wide range of fungal pathogens has been confirmed but, as with bacteria, some species are insensitive.
Dissecting Cf-4 and Cf-9 disease resistance gene specificity by domain
swaps and DNA shuffling
The tomato Cf-4 and Cf-9 genes confer resistance to the fungus Cladosporium fulvum through recognition of the Avr4 and Avr9 elicitors. Cf-4 and Cf-9 are 91% identical. Differences between the two proteins are found in domains A and B and their N-terminal leucine-rich repeats (LRRs) in domain C1, and consist of deletions, LRR copy number variation and amino acid polymorphisms. Thirty-two of the sixty-seven polymorphic amino acids are putative solvent exposed residues in the LRRs.
In order to determine which of the structural differences account for Cf-4 and Cf-9 specificity we have carried out domain swaps between Cf-4 and Cf-9. The chimeric clones have been tested in transgenic tobacco and tomato plants and/or Agrobacterium-mediated transient expression assays for their ability to induce an Avr-dependent hypersensitive response (HR). Gene shuffling (Crameri et al., Nature, 391:288-291) was carried out to increase the number of chimeras that could be analysed. This has enabled us to identify structural differences and amino acid residues that contribute to recognition specificity in Cf-4 and Cf-9. These include LRR copy number, sequence residues in domain B and in the central LRRs of domain C1, a region that exhibits hypervariability when homologues of Cf-4 and Cf-9 are compared.
We have recently used the gene-shuffling technology to shuffle homologues other than Cf-4 and Cf-9. These libraries will be screened for clones that confer a HR towards Avr4 or Avr9 in an attempt to evolve synthetic Cf-4 and Cf-9 genes.