British Society for Plant Pathology

BSPP Presidential Meeting 1997

Plant Pathology - Global Perspectives of an Applied Science

PH Gregory Prize Competition

Development of a PCR-based detection technique for Pyrenopeziza brassicae, causal agent of light leaf spot on winter oilseed rape (Brassica napus L. subsp. oleifera).
Simon J. Foster1, A.M. Ashby1 & B.D.L. Fitt2
1
Department of plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA.
2 IACR Rothamsted. Harpenden, Hertfordshire, AL5 2JQ

The heterothallic discomycete fungus Pyrenopeziza brassicae Sutton and Rawlinson (anamorph Cylindrosporium concentricum) is the causal agent of light leaf spot of brassicas. In particular, light leaf spot is considered to be one of the most damaging diseases of winter oilseed rape (Brassica napus L. subsp. oleifera) in the UK. The work described here represents the initial stages in the development of a polymerase chain reaction (PCR) based diagnostic technique that will be used to detect P. brassicae in infected plant tissues. Degenerate PCR primers, designed to amplify a region of DNA encoding the Sex Factor Induced (SFI1) protein, were shown to amplify a 700 base pair (bp) product from DNA extracted from a range of P. brassicae isolates of mating type MAT 1-2. This PCR product was not generated upon amplification of DNA from other fungal pathogens of oilseed rape and was shown to hybridise to 5kb (kilo base pairs) and 3kb Sal I fragments from P. brassicae isolates of mating type MAT 1-1 and MAT 1-2 respectively. Sequence analysis of the 700 bp PCR fragment enabled the design of fully homologous primers which facilitated the amplification of PR products from P. brassicae DNA only. These primers also differentiated between the two mating type of P. brassicae. These findings detailed here and discussed in relation to a light leaf spot forecasting system currently under development.


Production, separation, toxicity and metabolism of the solanapyrone toxins produced by the chickpea pathogen, Ascochyta rabiei
Khalid Hamid
Department of Biology, Darwin Building, University College London, Gower Street, London C1E 6BT

Ascochyta rabiei secreted the toxins solanapyrones A, B and C when grown on a medium consisting of Czapek Dox nutrients supplemented with cations. After filtering off the fungus, the toxins were partitioned into ethyl acetate and separated by flash chromatography on silica gel using 1. cyclohexane : dichloromethane : ethyl acetate 3:3:1 (v/v/v), 2. the same solvents but in equal proportions and lastly 3. ethyl acetate. Samples of the crude ethyl acetate fraction, obtained by partitioning culture filtrates, and pure compounds from flash chromatography were chromatographed on a C18 HPLC column with tetrahydrofuran : methanol : water 20.6 : 23.1 : 56.3 (v/v/v) in order to quantitate the toxins and determine their recovery.

The range of sensitivity of cells isolated from leaflets of 12 cultivars of chickpea to solanapyrone A varied 5-fold and solanapyrone A was 2 - 12 times more toxic than solanapyrone B, depending on cultivar. When shoots of chickpea were placed in solutions of solanapyrone A the stems lost their turgor and became shrivelled. In contrast, the stems of shoots placed in solanapyrone B remained turgid but the leaves became twisted and chlorotic. These symptoms, are typical of Ascochyta blight. Both compounds were metabolised by shoot cuttings, cells isolated from leaflets and by a protein preparation from shoots of the plants.


Relationship between height and resistance to fusarium ear blight in wheat
Alex J. Hilton1 , P. Jenkinson1, T.W. Hollins2 & D.W. Parry3
1
Crop and Environment Research Centre, Harper Adams Agricultural College, Newport, Shropshire TF10 8NB
2 Plant Breeding International, Maris Lane, Trumpington, Cambridge
3 Horticultural research International - East Malling, West Malling, Kent

The significance of morphological characters such as straw height, peduncle length, compactness of ear and angle of flag leaf on the development of Fusarium ear blight in eight cultivars of winter wheat was studied in a field trial in 1994/95. Straw height was shown to be significantly correlated to disease severity with taller cultivars such as Spark and Cadenza showing fewer symptoms of FEB compared with shorter cultivars such as Brigadier and Genesis. No other morphological character showed any significant correlation with disease.

To determine if this relationship was the result of genetic associations, cultivars of varying height were intercrossed, and the resulting F3 lines were assessed following artificial inoculation. Among random F3 populations, there was a clear tendency for tall straw and resistance to FEB to co-segregate. The effect of the individual dwarfing genes Rht 1 and Rht 2 on severity of FEB were studied in an artificially inoculated field trial in 1996/97 using a number of near-isogenic lines of Maris Huntsman. The presence of the dwarfing genes caused a significant increase in severity. These trials suggested that the relationship between disease and height is controlled either linked genes or genes with a pleiotropic effect.

The monitoring of humidity at ear height in both short and tall isogenic lines of Maris Huntsman showed no difference between lines in the shorter lines that could explain an increase in severity of FEB. It was concluded that the relationship between disease severity and height is controlled by linked genes and not by micro-climate effects.


Ultrastructure and composition of the cell surface of Colletotrichum lindemuthianum conidia.
Bleddyn Hughes1, R. Carzaniga2,3, R.J. O'Connell2 and J.R. Green1
1
School of Biological Sciences, University of Birmingham, Birmingham, B15 2TT
2 IACR - Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF; 3Istituto di Patologia Vegetale, Universita di Milano, Via Celoria 2, 20133 Milano, Italy

The spores of Colletotrichum lindemuthianum, causal agent of bean anthracnose, are coated with a fibrillar layer arranged perpendicular to the cell wall. This spore coat is stained by silver proteinate and removed after protease digestion, indicating the presence of glycoproteins. The monoclonal antibody UB20 was raised to germlings growing in vitro and recognises a carbohydrate epitope carried by glycoproteins on the spore surface (Pain et al., 1992). Western blotting is used to demonstrate that the glycoproteins recognised by UB20 are extracted from spores by hot water, SDS and by -mercaptoethanol at alkaline pH. Electron microscopy shows that the spore coat is also removed by these treatments. The major glycoprotein extracted from the cell is identified at 100 kD. Biotinylation of washed spores shows that this glycoprotein is located at the spore surface. Further characterisation of the glycoprotein demonstrates that it contains both O-linked and complex N-linked carbohydrate side-chains. High performance anion-exchange chromatography suggests that the carbohydrate moiety nay contain rhamnose. The 100 kD glycoprotein is currently being characterised using molecular biological techniques and a monoclonal antibody is being raised. Possible functions of the 110 kD glycoprotein will be discussed.

Reference
Pain et al. (1992). Physiological and Molecular Plant Pathology, 41, 111-126.


The role of saprophytic microflora in the development of fusarium ear blight of winter wheat caused by Fusarium culmorum.
Jo Liggit
Crop and Environment Research Centre, Harper Adams Agricultural College, Newport, Shropshire TF10 8NB

A number of fungicides were shown to give effective control of F. culmorum in vitro, moderately effective control of Fusarium ear blight (FEB) in the glasshouse, but poor control under field conditions. It as proposed that saprophytic microflora may interact with ear blight pathogens of wheat and contribute to the poor performance of fungicides against this disease. A glasshouse experiment and a series of experiments in vitro were conducted to determine the relationship between saprophytic microflora and Fusarium culmorum and to determine fungicide effects on Alternaria alternata, Botrytis cinerea, Cladosporium herbarum and Fusarium culmorum. Inoculation of winter wheat ears (cultivar Avalon) in the greenhouse with either A. alternata, B. cinerea or C. herbarum at GS 59 prior to inoculation with F. culmorum at GS 69 led to a decrease in the severity of FEB. The mean percentage of spikelets infected was reduced from 19% for ears which had been inoculated with F. culmorum alone to 4, 6 and 5% for ears which had been inoculated with A. alternata, B. cinerea and C. herbarum, respectively. In vitro, the mycelial growth of F. culmorum was reduced when inoculated opposite A. alternata, B. cinerea and C. herbarum. The antagonism was shown to be due to production of non-volatile and volatile antibiotics by the saprophytes showed differential sensitivity to the fungicides benomyl, chlorothalonil, fluquinconazole, flusilazole, flutriafiol, prochloraz, pyrimethanil and tebuconazole.

The possibility that fungicides differentially suppress saprophytic microflora on the ears of wheat leading to poor control of FEB in the field is discussed.


Identification and toxicity of Alternaria isolates obtained from cruciferous crops grown in Thailand
Preprame Pattanamahakul
Department of Biology, Darwin Building, University College London, Gower Street, London C1E 6BT

Fungi isolated from annular, necrotic leaf spots on leaves of cabbage, cauliflower, Chinese Kale and Choi-Sum, grown in northern Thailand, produced pigmented multicellular spores characteristic of the genus Alternaria. Comparison of the nucleotide sequences of the ITS2 region of the ribosomal DNA of eight of the isolates, two from each host plant, with that published for an authentic isolate of Alternaria brassicicola showed complete identity.

Filtrates from cultures grown on Czapek-Dox nutrients, supplemented with cations, were toxic to cells isolated from all four host plants. An isolate from cauliflower was the least toxic and one from cabbage the most. About half the activity was retained by a dialysis membrane and the remainder was diffusible and partitioned into ethyl acetate. Purification of the ethyl acetate fraction by solid phase extraction on C18 cartridges and thin layer chromatography on silica gel led to the isolation of two toxic compounds with characteristic UV spectra.