Brian Williamson

Botrytis cinerea: the cause of grey mould disease.

INTRODUCTION Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle attacking over 200 crop hosts worldwide. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. It has become an important model for molecular study of necrotrophic fungi. TAXONOMY Kingdom: Fungi, phylum: Ascomycota, subphylum: Pezizomycotina, class: Leotiomycetes, order: Helotiales, family: Sclerotiniaceae, genus: Botryotinia. HOST RANGE AND SYMPTOMS Over 200 mainly dicotyledonous plant species, including important protein, oil, fibre and horticultural crops, are affected in temperate and subtropical regions. It can cause soft rotting of all aerial plant parts, and rotting of vegetables, fruits and flowers post-harvest to produce prolific grey conidiophores and (macro)conidia typical of the disease. PATHOGENICITY B. cinerea produces a range of cell-wall-degrading enzymes, toxins and other low-molecular-weight compounds such as oxalic acid. New evidence suggests that the pathogen triggers the host to induce programmed cell death as an attack strategy. Resistance: There are few examples of robust genetic host resistance, but recent work has identified quantitative trait loci in tomato that offer new approaches for stable polygenic resistance in future. USEFUL WEBSITES http://www.phi-base.org/query.php, http://www.broad.mit.edu/annotation/genome/botrytis_cinerea/Home.html, http://urgi.versailles.inra.fr/projects/Botrytis/, http://cogeme.ex.ac.uk.

Botrytis : biology, pathology and control

Preface.- Acknowledgements.- Contributors.- 1: Botrytis spp. and diseases they cause in agricultural systems - an introduction Yigal Elad, Brian Williamson, Paul Tudzynski and Nafiz Delen.- 1 Introduction. 2. Geographical and ecological occurrence. 3. Variability and adaptability. 4. Quiescent, restricted and aggressive infection. 5. Molecular basis of host-parasite interactions. 6. References. 2: The Ecology of Botrytis on Plant Surfaces Gustav Holz, Sonja Coertze and Brian Williamson.- 1. Introduction. 2. Survival. 3. Inoculum production and dispersal. 4. Growth on plant surfaces. 5. Infection pathways on diverse plant organs. 6. Conclusion. 7. References. 3: Taxonomy and Genetic Variation of Botrytis and Botryotinia Ross E. Beever and Pauline L. Weeds.- 1. Introduction. 2. Taxonomy. 3. Botrytis cinerea. 4. Genetics of other species of Botrytis. 5. The future. 6. Acknowledgements. 7. References. 4: Approaches to Molecular Genetics and Genomics of Botrytis Paul Tudzynski and Verena Siewers.- 1. Introduction. 2. Generation of transgenic Botrytis strains. 3. Unbiased gene cloning systems. 4. Perspectives. 5. Acknowledgements. 6. References. 5: Morphology and Cellular Organisation in Botrytis Interactions with Plants Klaus B. Tenberge.- 1. Introduction. 2. Cytology and ultrastructure of Botrytis. 3. Imaging of infection. 4. Host response. 5. Conclusions. 6. Acknowledgements. 7. References. 6: Signalling in Botrytis cinerea Bettina Tudzynski and Christian Schulze Gronover.- 1. Introduction. 2. Ga subunits of hetrotrimeric G proteins. 3. cAMP signalling pathway. 4. MAP kinase pathways. 5. Genes of the Ras superfamily. 6. Calcineurin/cyclophilin A signalling. 7. Putative transmembrane receptor proteins. 8. Two-component signal transduction genes in Botrytis cinerea. 9. Further protein kinase encoding geneswith unknown function. 10. Conclusion. 11. References. 7: Extracellular Enzymes and Metabolites Involved in Pathogenesis of Botrytis Ilona Kars and Jan A.L. van Kan.- 1. Introduction. 2. Penetration of the host surface. 3. Killing of host cells. 4. Conversion of host tissue into fungal biomass. 5. Other enzymes potentially involved in pathogenesis. 6. Concluding remarks. 7. Acknowledgements. 8. References. 8: Botrytis cinerea Perturbs Redox Processes as an Attack Strategy in Plants Gary D. Lyon, Bernard A. Goodman and Brian Williamson.- 1. Introduction. 2. Hydrogen peroxide and other AOS. 3. Low molecular mass antioxidant molecules. 4. Perturbation of free radical chemistry as a result of Botrytis infection. 5. Production of oxalic acid. 6. Dynamics of iron redox chemistry. 7. Regulation of plant enzymes. 8. Botrytis-derived enzymes. 9. Generation of lipid peroxidation products. 10. Host signalling and programmed cell death. 11. Fungus-derived metabolites. 12. Conclusion. 13. Acknowledgements. 14. References. 9: Plant Defence Compounds against Botrytis Infection Peter van Baarlen, Laurent Legendre and Jan A.L. van Kan.- 1. Introduction. 2. Antimicrobial secondary metabolites. 3. Tolerance of Botrytis to antifungal metabolites. 4. Structural barriers and cell wall modifications. 5. Pathogenesis-related proteins. 6. Concluding remarks. 7. Acknowledgements. 8. References. 10: Phytohormones In Botrytis-Plant Interactions Amir Sharon, Yigal Elad, Radwan Barakat and Paul Tudzynski.- 1. Introduction. 2. Biosynthesis of plant hormones by B. cinerea. 3. Effect of plant hormones on B. cinerea and on disease development. 4. Conclusions. 5. Acknowledgement. 6. References. 11: Detection, Quantification and Immunolocalisation of Botrytis species Frances M. Dewey (Molly) and David Yohalem.- 1. Introduction. 2. Classical plating out method. 3. Immu

Functional analysis of H2O2-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable

SUMMARY The oxidative burst, a transient and rapid accumulation of reactive oxygen species (ROS), is a widespread defence mechanism of higher plants against pathogen attack. There is increasing evidence that the necrotrophic fungal pathogen Botrytis cinerea itself generates ROS, and that this capability could contribute to the virulence of the fungus. Two potential H(2)O(2)-generating systems were studied with respect to their impact on the interaction of B. cinerea and its host plant Phaseolus vulgaris. A Cu-Zn-superoxide dismutase gene (bcsod1) and a putative glucose oxidase gene (bcgod1) were cloned and characterized, and deletion mutants were created using a gene-replacement methodology. Whereas the Deltabcgod1-mutants displayed normal virulence on bean leaves, the Deltabcsod1 mutants showed a significantly retarded development of lesions, indicating that the Cu-Zn SOD-activity is an important single virulence factor in this interaction system. Whether dismutation of (fungal or host) superoxide, or generation of H(2)O(2) (or both), are important for pathogenesis in this system remains to be elucidated.

The adenylate cyclase (BAC) in Botrytis cinerea is required for full pathogenicity

SUMMARY The grey mould Botrytis cinerea is an economically important plant pathogen. Previously we found that null mutants of bcg1 encoding one of the two Galpha subunits of heterotrimeric GTP-binding proteins differed in colony morphology and showed reduced pathogenicity. To further understand the mechanisms involved in infection, we cloned the bac gene encoding adenylate cyclase, the enzyme that catalyses production of cAMP from ATP. The deduced protein sequence consists of 2300 amino acids, the ORF is interrupted by three conserved introns, and there is a high degree of similarity with the catalytic domains of other fungal adenylate cyclases. Gene replacement resulted in reduced vegetative growth and a morphology similar to that of bcg1 mutants. The wild-type (WT) colony morphology was partially restored by feeding exogenous cAMP. These bac mutants still had a low but constant level of cAMP, despite deletion of the complete catalytic domain of the enzyme. Conidia from bac mutants germinated, penetrated the leaves of Phaseolus vulgaris and caused spreading soft rot lesions (in contrast to bcg1 mutants), although these were slower to develop than in WT controls. Compared to the latter, the most striking difference was that no sporulation occurred on leaves inoculated with bac mutant conidia. These results confirm that the cAMP signalling pathway plays an important role in vegetative growth and pathogenicity in B. cinerea. On the other hand, a much stronger effect of bcg1 mutation on pathogenicity in comparison to the effects of bac mutations suggests that BCG1 controls at least one more signalling component other than adenylate cyclase, and that the cAMP signalling pathway is not the only one responsible for pathogenicity.

Botrydial is produced in plant tissues infected by Botrytis cinerea.

The fungal metabolite botrydial was detected for the first time in ripe fruits of sweet pepper (Capsicum annuum) wound-inoculated with conidial suspensions of Botrytis cinerea and also in leaves of Phaseolus vulgaris and Arabidopsis thaliana inoculated without wounding. This phytotoxin was produced in soft rot regions of the infection. In C. annuum, the most aggressive isolate produced the highest botrydial concentrations in planta. The levels of botrydial produced by this isolate did not correlate with the reported relative susceptibilities of four P. vulgaris genotypes. The results suggest that botrydial is a pathogenicity factor for this fungus, but not a primary determinant of pathogenicity.

Effect of humidity on infection of rose petals by dry-inoculated conidia of Botrytis cinerea

Cut flowers of rose cv. Scarlet Pimpernel were dusted with dry conidia of Botrytis cinerea from 10- to 14-d-old cultures and incubated at 15 °C in a range of humidities controlled to within 2% relative humidity (r.h.). Petals developed lesions within 24 h at, or above 94% r.h. of the air entering the controlled environment chamber containing rose flowers in the absence of water droplets, and necrosis was severe after 48 h. Petals were cleared, stained in aniline blue and viewed by fluorescence microscopy to show that conidia germinated to form only short germ tubes. They lacked appressoria or any other complex infection apparatus and colonized petals extensively within 48 h. Low temperature scanning electron microscopy confirmed that the conidia germinated in the absence of surface water and penetrated the epidermis without the formation of appressoria or evident external surface changes in the cuticle around germ tubes at the point of penetration of host cells. In freeze-fractures of infected petals, the apices of fungal hyphae caused no apparent deformation of host cell walls during penetration of, or emergence from, the mesophyll cells, indicating that cell wall-degrading enzymes are secreted and retained at the hyphal tip.

Purification and characterization of four polygalacturonases from Botrytis cinerea

An isolate of Botrytis cinerea produced four polygalacturonases (PGs) in a modified Czapek-Dox liquid medium containing citrus pectin. Purification involving liquid-phase isoelectric focusing, ion-exchange chromatography and concanavalin A-affinity chromatography revealed two endo-PG isozymes (endo-PGI and endo-PGII) with pI values of 8·8 and 4·9 and two exo-PGs (exo-PGI and exo-PGII) with pl values of 4·9 and 3·5 in 10-day-old cultures. Endo-PGI and II both had apparent M r values of 36 kDa; exo-PGI and II had M r values of 65 and 70 kDa respectively. Each protein consisted of a single polypeptide chain. Exo-PGI and II were shown to be glycoproteins by their affinity to concanavalin A. The temperature optima for endo-PGI, endo-PGII, exo-PGI and exo-PGII were 37, 34, 48 and 50 °C respectively, and all the isozymes showed optimal pH values between 4·0 and 5·0.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea

Abstract. The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

Botrytis cinerea Perturbs Redox Processes as an Attack Strategy in Plants

Plant tissues and pathogen inoculation. Leaves of intact plants of P. vulgaris and A. thaliana were inoculated with 14-day-old conidial suspensions of B. cinerea isolate B05.10 under conditions that gave rise to spreading soft rots ; other work used detached fruits of C. annuum inoculated with another isolate . Samples for analysis were taken from within lesions, at the lesion margins and at varying distances into uninfected tissue, and compared with samples from non-inoculated controls held under the same conditions.

An NMR microscopic study of grape (Vitis vinifera L.)

SummaryMature healthy grape berries and berries wound-inoculated with the fungusBotrytis cinerea were examined by1H NMR microimaging using 2D and 3D spin echo and gradient echo procedures. These NMR images were compared with representations obtained by conventional histology, where possible using the same specimens. 3D imaging datasets from excised seeds were reconstructed by surface rendering and maximum intensity projection to allow interpretation of their internal structure. T2-weighted spin echo images revealed the major features of the pericarp, septum and loculi of whole berries. T1-weighted images were less discriminatory of parenchyma tissues in the fruit but revealed the endosperm in seeds as a chemically shifted feature. A non-invasive study by T1-weighted spin echo NMR imaging of infection byB. cinerea over a 6-day period showed that the disease spread throughout the exocarp but failed to spread in the mesocarp, a result confirmed by histological examination of the same specimen. Surface rendering of 3D datasets of excised seeds revealed the two ruminations of the endosperm and the distal location of the chalaza. The position of the embryonic axis was revealed in T2-weighted maximum intensity projections. This noninvasive study revealed the need to apply a range of imaging techniques and parameters to visualise the structural features of the different parts of the grape berry.