BSPP Presidential Meeting 1999

Biotic Interactions in Plant-pathogen Associations

Competition Posters

Preliminary studies on blackspot of roses (Diplocarpon rosae).
Alefayah Ali, Avice Hall, * Charles Lane, Roger Cook, Paul Beales**, Paul Cannon***

* Environmental Sciences, University of Hertfordshire, College Lane, Hatfield AL10 9AB
** Central Science Laboratory, Sand Hutton, York, N. Yorks YO41 1LZ
*** CABI Biosciences, Bakeham Lane, Egham, Surrey TW20 9TY

Rose blackspot causes considerable damage to roses in commercial production (exports alone are worth 669,000 in 1997) and to roses grown by amateurs in their gardens. Some commercial producers spray up to 40 times a year, and many amateurs also spray fungicides with great frequency. Amateur use of pesticides has already been banned in Germany, and this ban is likely to become EU wide in the future. It is in this context that MAFF is funding a 3 year collaborative project with Environmental Sciences, University of Hertfordshire, CSL, York, and CABI Bioscience at Egham.

The overall aims of the project are:

1) To obtain 100 isolates of D. rosae from throughout the UK and also from at least 20 locations overseas.
2) To develop a standard pathogenicity test for evaluating new cultivars and fungicides.
3) To determine the trade pathways for roses entering the UK.
4) To investigate the cultural characteristics, physiological types and pathotypes of the Diplocarpon rosae isolates.
5) To ascertain the level of fungicide resistance / tolerance of selected isolates.
6) To assess the feasibility of in vivo identification of pathotypes with molecular diagnostic tools.
7) To ascertain the environmental factors favourable to disease development and the prevalence and role of the sexual stage on fallen overwintering leaves.

The work reported in this poster particularly addresses aims 1,2,3,4and 7.

Over 100 isolates have been collected from the UK, and the collection was 'stratified' in that isolates were obtained from different parts of the UK, and from different types of roses (climbers, ramblers, hybrid teas, species roses, etc.). Overseas isolates were obtained by CSL York. These isolates were stored both in vitro and in vivo, the latter on leaf discs of the cv. Frensham. Preliminary results are available for the morphological characteristics of isolates in vitro .A standard pathogenicity test has been developed, and differential cultivars selected and pathogenicity tests started. Epidemiological work has also started, with particular emphasis on the environmental conditions in the winter, and on the build up of the epidemic in the spring.

Association of dsRNA with effects on growth and virulence in Verticillium fungicola infecting cultivated Mushroom.
Angsana Akarapisan, R.G.T. Hicks and J.M. Clarkson.

Department of Biology and Biochemistry, Faculty of Science, University of Bath, Bath BA2 7AY, UK.

Verticillium spp. remain one of major threats to Agaricus bisporus production throughout the world. Little is known about the pathogen diversity or the interaction between pathogen and host. Symptoms are varied and are thought to depend on a number of factors such as developmental stage, the time of infection and genetic variability of the host and pathogen. Six geographically different strains of Verticillium spp. were assayed for the presence of extrachromosomal nucleic acid elements. Two strains were found to harbour double-stranded RNA (dsRNA) elements. Five dsRNA segments were found in V. fungicola strain V7-3 with apparent molecular weights about 1.1-2.4 Kb. Only one dsRNA segment was detected in V. psalliotae strain V5-2 with apparent molecular weight 0.7 kb.

Using a phosphate buffer extraction protocol, isometric particles approximately 20 nm in diameter were purified from the mycelial tissue of strain V7-3 (+dsRNA). Initial analysis suggests that the viral capsid consists of one associated major protein with a molecular weight of ca. 36 kDa. Comparison of isogenic strain V7-3 (+dsRNA) and V7-3h (-dsRNA, heat treatment) indicated that significantly reduced growth rate, dry weight and sporulation was associated with the presence of dsRNA. Virulence experiments, using mushroom fruit bodies determined that dsRNA associated with V7-3 strain significantly reduced virulence. Complementary DNA clones from V7-3 dsRNA were partially sequenced and shown to contain conserved amino acid motifs typical of virus RNA-dependent RNA polymerases (RDRP).

Pathogenicity and crucifer isolates of Verticillium dahliae
A. Collins, 1D. Barbara, 2D. Parry & 3S. Edwards

1Horticulture Research International, Wellesbourne, Warwickshire
Horticulture Research International, East Malling, Kent
Harper Adams University College, Newport, Shropshire

V. dahliae and V. albo-atrum are important soil-borne plant pathogens causing vascular wilts in a wide range of crops throughout the world. The majority of these isolates are haploid and do not infect cruciferous plants. However, some isolates of V. dahliae which produce long spores and have a high DNA content are major pathogens of such plants. These isolates have been suggested to be natural interspecific hybrids, which probably arose through parasexual hybridisation between V. dahliae and V. albo-atrum. This has resulted in the evolution of amphihaploid isolates with a novel pathogenicity. The long-term goal of the project is to understand the molecular basis of this novel pathogenicity. Molecular approaches are now being used to investigate the interrelationships of Verticillium isolates and the phylogeny of the plant pathogenic species. The initial results of this work will be presented.

Sequence analysis of Furoviruses of cereals from China and Europe.
Aipo Diao
and M.J.Adams

Plant Pathology Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ,UK

Soil-borne wheat mosaic virus (SBWMV), the type member of the genus Furovirus, has been reported from most winter wheat growing regions of the world including the USA, Brazil, France, Italy, Egypt, Japan and China. We report here the complete sequences of Oat golden stripe virus (OGSV) and two wheat furoviruses isolates from France and China that have been named Chinese wheat mosaic virus (CWMV) and European wheat mosaic virus (EWMV) respectively.

Yam viruses of the South Pacific Islands.
Bndicte Lebas
, Ed Canning, Lawrence Kenyon and Susan Seal

University of Greenwich, Natural Resources Institute, Pest Management Dpt, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK. 

In the South Pacific Islands, yams are a popular and important food crop cultivated for their tubers, which are rich in carbohydrates. The two dominant cultivated yam species in the South Pacific are Dioscorea alata and D. esculenta. Little is known about the diversity and variability of viruses infecting yams in the South Pacific Islands, which is a hindrance to the safe movement of germplasm within and outside of this region.

Leaf samples (69 and 229 accessions respectively from the field genebanks in Fiji and Vanuatu) have been tested by ELISA using antisera directed at yam mosaic virus (YMV), D. alata potyvirus (DaV), D. alata badnavirus (DaBV), D. bulbifera badnavirus (DbBV), D. latent potexvirus (DLV), D. dumetorum potyvirus (DdV) and cucumber mosaic virus (CMV). DaV was found to be the most common virus (74% of samples), followed by CMV (55%), DdV (12.4%), DabV(5%), and DLV (4.4%). YMV was not detected in any of the samples from the South Pacific. The sensitivity and specificity of the serological tests have been confirmed by electron microscopy and PCR. A reverse transcription (RT)-PCR for the detection of DaV has been optimized. However, some ELISA-positive samples were not detected by a specific RT-PCR, and vice versa. In addition, a number of ELISA-positive DaV samples are proving difficult to amplify by PCR using degenerate general potyvirus primers. These preliminary results suggest some variability in the DaV genome. PCR detection of badnaviruses has also been optimized using oligonucleotide primers for African strains. However, none of the badnavirus-positive ELISA samples from Fiji produced an amplification product with these primers, suggesting sequence differences in the virus genomes from the different regions.

Plant tissue culture from nodal cuttings is being used for the maintenance of yam germplasm, and chemotherapy will be tested for the production of virus-free plants. Reliable and robust indexing procedures will enable the safe movement of yam germplasm, while the use of virus-tested plants should result in increased yam yields.

Identification and characterisation of Arabidopsis thaliana expressed sequence tags representing novel genes for chitinases and thaumatin-like proteins.
N. N. Ludidi, W. Hide* and D. J. G. Rees

Department of Biochemistry and the South African National Bioinformatics Institute*, University of the Western Cape, Bellville, 7535, South Africa.

Chitinases and thaumatin-like proteins (TLPs) are plant antifungal proteins that play a role in plant defence against fungal pathogens. We have analysed sequences in protein, nucleic acid and expressed sequence tag (EST) databases to identify ESTs in the EST databases that represent novel genes encoding chitinases and TLPs. We are currently performing experiments aimed at finding out the copy numbers and patterns of expression of these genes; and expressing a full length cDNA clone in E.coli, encoding a TLP, to examine the antifungal activity of the expressed protein. This study may have significance in the genetic engineering of plants for fungal disease resistance.

Towards molecular detection of strawberry crinkle virus.
Karin Posthuma
, Yiguo Hong & Tony Adams

Horticulture Research International, East Malling, West Malling, Kent ME16 6BJ, UK.

Strawberry crinkle virus (SCV), a plant rhabdovirus transmitted by the strawberry aphid Chaetosiphon fragifolii Cockerell, decreases strawberry yield and fruit quality. The routine detection method for SCV is grafting, as no laboratory test is available. In this project we try to develop a detection method based on the polymerase chain reaction (PCR). PCR requires information on the sequence of SCV. To obtain this information three subtracted libraries were constructed. Screening of these libraries revealed many putative plant defence related genes but virus sequence information has hitherto not been obtained.

Resistance to turnip mosaic virus in Brassica.
Sara L. Hughes1,2, R.L. Rusholme3,4, D.J. Lydiate4, M.J. Kearsey2 and J.A. Walsh1

See P H Gregory Prize Competition abstracts

Virulent (V1) and avirulent (V2) isolates of Verticillium albo-atrum against a resistant cultivar of Medicago sativa
Mingguo Tang
, Murat Dikilitas, C.J.Smith

School of Biological Sciences, University of Wales, Swansea, Singleton Park Swansea SA2 8PP, Wales, UK. 

Kabul, a resistant cultivar of Medicago sativa, was inoculated with both virulent (V1) and avirulent (V2) isolates of Verticillium albo-atrum. When it is inoculated with the spore suspension of fungi (1x107) with root-dipping method, only V1 had an affect on Kabul on height to some degree. But no significant visible symptoms were observed of the disease. When the inoculation is made with the isolate V2 no significance of height reduction and symptoms were found statistically important. Inoculated plants with V1 also showed tyloses, a characteristic response to the disease. Elicitors from V1 and V2 induced increase in PAL activity and phytoalexins accumulation in leaves tissue of Kabul, V1 induced higher PAL activity but less phytoalexin accumulation was observed. However in suspension cells of Kabul, V1 elicitor induced higher oxidative burst than that of V2, V1 induced greater increase of PAL activity, higher accumulation of phenolic polymer and phytoalexins. V1 also induced higher GST activity and glucanase activity. We concluded that, to resistant cultivar of plants, the behaviour of virulent and avirulent pathogen was similar except their extent being affection.

The effect of inoculation methods and temperature on disease development inoculated with Verticillium albo-atrum on tomato and lucerne plants
Murat Dikilitas
, C.J. Smith

School of Biological Sciences, University of Wales, Swansea,, Singleton Park Swansea SA2 8PP, Wales, UK. E-Mail:

The effect of temperature and inoculation methods on tomato and lucerne plants inoculated with Verticillium albo-atrum were studied. In 23C the effect of pathogen was severe and isolation was made from all the inoculated plants. When the temperature rose to 27C development of the disease was retarded while around 30C effects of the disease were not observed. In 30C, only one isolation was made from the infected tomato plants. Pathogenicity experiments also showed that cut-stem and wound inoculation methods were less effective than a root-dipping inoculation method with lucerne plants. Tomato plants inoculated with the fungus using either method were affected and showed severe disease symptoms such as epinasty, wilting, chlorosis and adventitious roots on the lower part of the stem. When stems were cut longitudinally dark-brown discoloration characteristic of the disease was evident. Plants also showed other responses to the disease, such as the presence of tyloses and lignitubers.