BSPP Presidential Meeting 2000

Plant-pathogen Interactions:
Understanding Mechanisms of Resistance and Pathogenicity for Disease Control

Offered Poster Abstracts - I

Fungicidal resistance to Diplocarpon rosae
Ali, A & Hall, A M
University of Hertfordshire, College Lane, Hatfield, Herts AL10 9AB Tel: 01707-285092

Roses are among the most economically important ornamental plant species in the UK, with exports alone worth 669,000 in 1997. One of the most severe diseases of field grown roses is Rose Blackspot, caused by the host specific facultative parasite Diplocarpon rosae (Horst, 1983). Infections can be controlled by regularly spraying leaves with a broad range of protectants and systemic fungicides which are available to both commercial growers (e.g. Captan, dichlofuanid, mancozeb, myclobutanil and triforine) and amateurs (bio Spraydex, Nimrod-T and Roseclear 2). Due to the repeated and continual use (up to 40 times a year) of fungicides with similar modes of action, the selection pressure on the fungus is greater and so the development of fungicide resistance in the fungal population is a possible response.

The aim of the study is to screen selected isolates of D. rosae (collected from different rose varieties and also from a wide geographical area) for their sensitivity (resistance/tolerance) to a range of active ingredients that are used to control this disease.

A range of methods has been employed to screen the different active ingredients. Detached leaves were sprayed with the systemic fungicide ingredients and then inoculated with an isolate one week later. Poison disk and poison plate methods were used to assess the sensitivity of a range of contact fungicides.

Preliminary results show that the isolates are still sensitive to the active ingredients when tested using the poison disk method.

As the presence of fungicidal resistance would have an impact for control, the results of this study will be used to inform commercial and amateur growers and ultimately develop an integrated management plan.

Interactions between host roots and plant-parasitic nematodes
M.R. Armstrong, B. Banks, P. Birch, J. Jones, M.S. Phillips, J. Wishart and V.C. Blok
Unit of Mycology, Bacteriology and Nematology, Scottish Crop Research Institute, Invergowrie, Scotland DD2 5DA

Nematodes are economically important pests both in the UK and throughout the rest of the world. Sources of readily exploitable host resistance are limited. We are using differential molecular techniques (cDNA AFLPs and suppressive subtractive hybridisation (SSH)) to identify genes involved in interactions between various potato and tomato genotypes and nematode parasites which may offer opportunities for novel and stable resistance to several EU quarantine organisms.

To undertake these studies, root systems were generated either directly from seed, potato shoots taken from tubers or from in vitro propagated plants. For synchronized infections, roots were inoculated with juvenile nematodes and invasion allowed to proceed for 24h. Root tissues were collected at various time points, RNA extracted, cDNA synthesized and used in SSH or cDNA AFLP analyses.

Host genes induced during the susceptible responses to Globodera pallida, G. rostochiensis and Meloidogyne chitwoodi have been identified using both techniques. Some have previously been identified in stress responses and in wounding by pathogens whilst others are common to response pathways induced by aerial pathogens.

Nematode genes have been identified in infected root material using SSH. Resistant and susceptible potato genotypes infected with Globodera rostochiensis were compared 24h after infection. In the resulting library, 60% of sequences were of nematode origin. This approach thus shows promise for investigating nematode gene expression during invasion, induction of the susceptible response and development in the feeding stages, which have thus far been difficult to study in vivo.

These techniques are also being used to compare avirulent and virulent nematode populations of Meloidogyne and Globodera spp.. Induction of the host resistant response differs with different combinations of host and nematode genotypes. Some host responses are rapid whereas others have a delayed response. Where the response is rapid, direct comparison of juvenile nematodes is being conducted. However, the SSH techniques is being used for gene expression analysis in planta where the host resistant reaction is delayed.

Funding from EU PL98-4235 (No Nematode), QLK5-CT-1999-01501 (Nonema), QLRT-1999-1462 (DREAM), and the Scottish Office Agriculture, Environment and Fisheries Department is gratefully acknowledged.

Building physical maps of Erwinia carotovora subspecies to compare their genomic organisation and to identify novel pathogenicity and host range-related genes
Avrova A1
, Bell KS1, Waugh R2, Milbourne D2, Toth IK1, Dellagi A1, De Jong W2, Hyman L1, Bryan G2 and Birch PRJ1
Unit of Mycology, Bacteriology and Nemotology1 and Unit of Genomics2, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

Erwinia carotovora subspecies atroseptica (Eca) and Erwinia carotovora subspecies carotovora (Ecc) are commercially important potato pathogens. Both bacteria secrete a wide range of plant cell wall degrading enzymes but while the host range of Eca is restricted to potato, that of Ecc is much wider. Although the molecular basis for these phenotypic differences is unknown, the recent discovery of a type III secretion system (hrp cluster) in the soft rot erwinias suggests that their pathogenicity and host range are more complex than previously thought.

We have created Bacterial Artificial Chromosome (BAC) libraries of Eca and Ecc and are using AFLP fingerprinting and a hybridisation strategy to generate complete physical maps of the genomes. For hybridisation, a selection of genes/sequences are being used as probes: Erwinia carotovora genes from international databases including known or putative pathogenicity genes, differentially expressed Eca genes (obtained using cDNA-AFLP), and end sequences of the Eca BAC clones including Eca-specific sequences. These Eca-specific sequences include matches to a putative integral membrane transport protein from Escherichia coli and a periplasmic linker protein from Pseudomonas putida. Regions of interest, including the hrp cluster and putative avirulence genes, have been identified and are currently being studied in more detail.

A comparison of the Eca and Ecc physical maps, as expected, shows the genomic organisation of the two subspecies to be highly related, although the position of some genes do differ between the genomes. The Eca and Ecc physical maps are also being compared with the fully sequenced E. coli genome to examine similarities in the genomic organisation of these closely related plant and human pathogens.

Biological properties of two Moroccan isolates of barley yellow dwarf virus-PAV
B. Bencharki1 and M. El Yamani2
Universit of Hassan 1st, Faculty of Sciences and Technology, P.O. Box 577 Settat, Morocco; e-mail: 2Centre Aridoculture INRA-Settat , Morocco.

Background and objectives
One the most common field cereal viruses in Morocco is the barley yellow dwarf virus (BYDV), a Luteoviridae with a wide host-range, particularly affecting gramineacous plants. BYDV is persistently transmitted by various aphid species of which Rhopalosiphum padi and Sitobion avenae appear to be the most important natural vectors. The BYD disease is caused by a complex of at least five viruses of which BYDV-PAV has a large geographical distribution and occurs at high incidence. The observation of plant-pathogen interactions revealed a high level of variation within BYDV-PAV. Based upon symptom expression on barley cultivars of the Moroccan BYDV-PAV isolates and their differences in the coat protein coding sequences showed that isolates were separated into two different clusters CPI (moderate) and CPII (severe) [1]. However, replication and spread of these two clusters have not yet been investigated. In this research, we investigated concentration and its evolution, within the two clusters for three barley genotypes and related the behavior to symptoms.

Results and conclusion
The evolution of the concentration of isolate CPI MA9501 (moderate) and CPII MA9514 (severe) was investigated in barley. We have used two resistant genotypes 80-81-BQCB-10 and Atlas 68, and a sensitive one Atlas 57. The concentration of the virus has been estimated by DAS-ELISA at different periods after inoculation. The resistant genotypes significantly reduced virus accumulation in aerial part of the plant. However, the level of resistance varied according to the genotype and isolate of the BYDV-PAV. When comparing resistant and sensitive genotypes, we noted that the concentration of the virus in the resistant plant leaves is lower than sensitive genotype. Moreover, the isolate MA9501 is less concentrated than MA9514. The isolates of the BYDV-PAV reached their maximal concentration in the plants two weeks after inoculation. Four weeks after inoculation, reduction of the viral concentration in the resistant genotypes compared to the sensitive ones for the MA9501 was 48% and 46%, in 80-81-BQCB-10 and Atlas 68, respectively. For the isolate MA9514, reduction in the accumulation was 64% and 54%, respectively.

The transmissibility of BYDV-PAV isolates by different sub-populations of R. padi and S. avenae was also examined after 4 and 48-hr acquisition access period. Especially, isolate MA9514 was more efficiently transmitted compared to the MA9501 isolate by all aphid sub-populations.

The simultaneous presence of the two viral clusters in host was not yet investigated, but could have various and unpredictable consequences in terms of symptomatology.

[1] Bencharki, B., Mutterer, J., El Yamani, M., Ziegler-Graff, V., Zaoui, D., and Jonard, G. 1999. Annals of Applied Biology 134: 89-99.

Identification and differentiation of Erwinia carotovora subspecies using 16S-23S ribosomal spacer PCR-RFLP and AFLP
Anna O Avrova
, Lizbeth J Hyman, Rachel L Toth and Ian K Toth
Unit of Mycology, Bacteriology and Nematology, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK.

The soft rot erwinias cause substantial crop losses world-wide, especially on potato. Understanding the relationships between these closely related pathogens is important for accurate disease diagnosis, pathogen detection and epidemiological analyses. In this study two complementary molecular approaches, PCR-RFLP of the intergenic 16S-23S ribosomal spacer region (ISR-RFLP) and Amplified Fragment Length Polymorphism (AFLP), were used to provide a rapid means of identifying E. carotovora subspecies and E. chrysanthemi, and to investigate genetic variation within the soft rot erwinias. ISR-RFLP, gave unique banding profiles for these pathogens, providing rapid and unequivocal identification within two days and allowing identification of strains previously classified as "atypical". AFLP increased the level of discrimination still further, and was used to examine the relatedness of species/subspecies at the molecular level.

The Erwinia amylovora type III secretion chaperone DspB is necessary to stabilize DspA production
Barny Marie-anne and Gaudriault Sophie
Laboratoire de pathologie vgtale, UMR 217 INRA/INA-PG/Universit Paris VI, 16 rue Claude Bernard, 75231 Paris cedex 05

Erwinia amylovora is a Gram negative bacteria responsible for fire blight, a necrotic disease affecting plants of the Rosaceous family. E. amylovora virulence is dependent on a functional type III secretion system. To date, four proteins have been shown to travel through this secretion system, HrpN, HrpW, HrpA and DspA (also called DspE). Cotranscribed with dspA, dspB (also called dspF) encodes a small acidic protein sharing similarities with the type III secretion chaperone described in animal system. Secretion chaperones assist specifically the secretion of one or two type III-secreted proteins. Here, we show that DspA was not secreted in a dspB background while other known type III-secreted proteins (HrpN, HrpW, HrpA) remained secreted to wild-type level. Therefore, DspB acts as a specific DspA chaperone. Further analysis showed that DspA was not detected in a dspB mutant background. Actually, expression of a dspA::lacZ translational fusion was abolished in a dspB background while expression of a dspA::uidA transcriptional fusion was slightly enhanced. Far western blot experiments demonstrated a physical interaction between DspA and DspB. All these results show that DspB acts downstream of dspA transcription and is necessary to stabilize the DspA protein production before secretion.

Molecular mapping of the Rph7.g leaf rust resistance gene in barley (Hordeum vulgare L.)
S. Brunner, B. Keller and C. Feuillet
Institute of Plant Biology, University of Zrich, Zollikerstr. 107, CH-8008 Zrich, Switzerland

In many temperate areas of the world, leaf rust is becoming an important disease of barley. In the last decade, new races of Puccinia hordei G. Otth have emerged which are virulent against the so far most effective race-specific resistance genes, such as Rph7. Marker-assisted selection greatly facilitates the pyramidization of two or more resistance genes in a single variety to achieve a more durable resistance. Such a strategy requires the development of efficient and reliable markers. Here, we have developed a linkage map and found RFLP markers closely linked to the Rph7.g resistance gene on chromosome 3HS of barley. The receptor-like kinase gene Hv3Lrk that maps at 3.2 cM from Rph7.g was used to develop a PCR-based marker by exploiting a single nucleotide polymorphism. This marker was detected in 11 out of 12 (92%) barley lines having Rph7 and represents a valuable tool for marker-assisted selection. In addition, the identification of markers flanking Rph7.g provides the basis for positional cloning of this gene.

Keywords: Barley, leaf rust, marker-assisted selection, resistance gene, Single nucleotide polymorphism.

Analysis of Differential Gene Expression in Ralstonia solanacearum using a cDNA-AFLP approach
Kirsty Bryant and Mark Bailey
NERC Centre for Ecology and Hydrology, CEH-Oxford, Mansfield Road, Oxford, UK.

The causative agent of potato brown rot and bacterial wilt, Ralstonia solanacearum, results in serious world-wide economic losses, particularly in the tropics. In the last decade, however, the incidence of bacterial wilt in potatoes grown in Northern Europe has increased presenting an interesting epidemiological puzzle. Is the occurrence due to change in agricultural practice or the emergence of a novel bacterial variety better adapted to cooler conditions? Our aim is to look at the fate and survival of this pathogen in European soils and water systems. One approach is to look at how the pathogen copes with different individual stresses, e.g. cold temperatures or dessication. Comparison of differential gene expression under different environmental conditions may provide insight for answering such questions.

The analysis of prokaryotic gene expression by cDNA-AFLP was first described by Dellagi et al. (2000)1, using Erwinia carotovora as a model system. We have applied this method to Ralstonia solanacearum. For any study of gene expression, it is desirable to synthesise cDNA representative of the whole transcriptome. Following the selection of appropriate 11mer primers (Fislage et al., 1997), designed to anneal to conserved regions of bacterial genes, a combination of ten primers was used to synthesise cDNA. Amplified fragment length polymorphism (AFLP) analysis of cDNA was undertaken to compare the fingerprint patterns of the transcriptome of bacteria grown under different environmental conditions. Novel fragments were identified and sequenced. Northern analysis was used to confirm that differentially amplified cDNA fragments are derived from differentially expressed genes.

To illustrate the utility of this comparative approach, cDNA was extracted from R. solanacearum grown in either LB broth or 10% (w/v) TS broth + sucrose. Specific PCR amplifications with primers designed to known differentially expressed R. solanacearum genes (epsC and phcA) confirmed the expression of both genes, thus suggesting that the cDNA may have good genome coverage. Optimisation of the method for use with R. solanacearum included comparison of different RNA extraction methods, different Taq polymerases, and primer design / combinations for differential display PCR. Imposed stress conditions tested to date include osmotic stress, nutrient limitation, (Halverson and Firestone, 2000), novel carbon sources e.g. potato dextrose agar, and growth on potato discs.

Transcripts isolated from LB vs 10% TS broth, and from minimal media with different equivalent matric potentials (-0.15 MPa vs 0.5 MPa) have been sequenced. By BLAST sequence analysis, five putative ORFs, with significant homology to bacterial (E. coli and B. subtilis) proteins, have been detected, including a B. subtilis protein with a possible osmotic protection function. These are being further characterised using RT-PCR and Northern hybridisations.

1 Dellagi, A., Birch, P. J. R., Helibronn, J., Lyon, G. D. and Toth, I. K. (2000) Microbiol. 146: 165-171

The distribution of avr / vir genes in Pseudomonas syringae legume pathogens
Dianne Butcher
52, Chandos Road, Rodborough, Stroud, Glos. GL5 3QZ

A pathogenicity island (PAI) containing virulence (vir) and avirulence (avr) genes on a 154 kb native plasmid from the phytopathogen, Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, was previously identified. Curing of this plasmid resulted in loss of virulence and the initiation of a hypersensitive response in previously susceptible bean cultivars. It was established that a 30 kb region on this plasmid, which contained known avirulence genes (avrD, avrPphC and avrPphF), also contained several putative genes (virPphA, ORF2, ORF3, ORF4 and tnp), which control virulence, along with a transposase gene. possession of this 30 kb region by genomic clones was responsible for restoration of virulence in the native strain (R.W. Jackson et al., 1999: Proc. Natl. Acad. Sci. USA 96:10875). The presence of homologues of the genes avrD, avrPphC, avrPphF, virPphA, ORF2, ORF3, ORF4 and tnp, was investigated in the related pathovar, Pseudomonas syringae pv. pisi. PCR and hybridization studies revealed the absence of virPphA, ORF2 and ORF3. ORF4 and tnp were present, but located on the chromosome. The significance of this pattern of gene distribution will be considered.

Pathogenicity and Resistance in Xanthomonas blight of cassava
Cooper, R.M., Kemp, B., Day, R., Gomez-Vasquez, R. and Beeching, J.R.
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK,

Resistant genotypes offer the only durable, practicable means of controlling, X. axonopodis pv manihotis (Xam) but the nature of defence of this key, staple crop is unknown; HR is not expressed to Xam and resistance, based on polygenes, is incomplete and dependent on environment and pathogen inoculum level. Rapid generation of reactive oxygen species (apparently via superoxide) occurred in suspension cells to diverse elicitors (but not to bacterial LPS) and to bacteria in leaf cells in response to incompatible bacteria but not to Xam. HR in leaves was preceded by superoxide production and was not affected by nitric oxide inhibitors. Subsequently, defence-related genes were expressed in elicited suspension cells including PAL (after 30 min, max 9-12h), HRGP and peroxidase (after 12h, max 48h) whereas catalase was rapidly down-regulated. AFLP analysis is revealing genes new to cassava; of 78 transcript-derived fragments ca. 75% were up-regulated and 25% down-regulated. Preformed putative defences include copious latex production which contains protease, 1,3 glucanase and lysozyme activities. The major phenolics are scopoletin, esculetin, ferulic acid and quercitin but these have relatively weak antifungal activity (enhanced after oxidation by peroxidase) and no toxicity to Xam.

Pathogenicity determinants of Xam are being investigated by disruption of a gum biosynthesis gene (EPS is produced copiously in planta) and a pel gene (pectate lyase appears as a single isoform); a putative toxin, MCPA, does not appear to contribute to infection.