Biocontrol of Botrytis-incited diseases has been extensively investigated over the last 50 years. This chapter reviews the literature on microbial control of Botrytis species with a particular emphasis on the importance of gaining a full understanding of the biological and ecological attributes of a biological control agent as a means to developing appropriate strategies for its effective use under commercial cropping conditions. The key microbial genera that have shown greatest potential for Botrytis disease control include the filamentous fungi Trichoderma, Gliocladium and Ulocladium, the bacteria Bacillus and Pseudomonas and the yeasts Pichia and Candida. Commercial success has been achieved in glasshouse and post-harvest environments where stable environmental conditions allow greater control over the application of the biocontrol agent and expression of its biological activity. Considerable progress has also been made in achieving more consistent biocontrol under field conditions, particularly in vineyards, but the complexities of the plant, microbe, environment interaction and its inherent variability will always pose a severe challenge to achieving effective and consistent field biocontrol. In recognising this, current research aims to define more clearly the biological and economic barriers that limit biocontrol efficacy and future research should focus on the strategic integration of biocontrol systems with other cultural, chemical and genetic methods to provide more sustainable disease control.

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.

Fertile complexes (individual reproductive units of ovulate cones) of three Prumnopitys species and Afrocarpus falcatus (Podocarpaceae) were subjected to histological examination and non-destructive NMR imaging. The latter technique allowed the display, frame-by-frame analysis and electronic 'dissection' of internal structures such as the number and courses of vascular traces and resin canals and their morphology. Characters of these internal structures distinguished all three Prumnopitys species from each other and thus were shown to be taxonomically diagnostic. Fertile complexes of Prumnopitys andina and P. taxifolia were most similar, possessing simple vascular traces and few unbranched resin canals. Those of P. ferruginea were very different and possessed an interconnected network of resin ducts within the sarcotesta. These findings are congruent with relationships inferred from molecular phylogenetic studies, in which two subclades were recovered within Prumnopitys. The anatomy of the female fertile complexes of Afrocarpus falcatus was very distinct from all Prumnopitys species analysed. Its most distinctive feature was the existence of a complex network of radial vascular strands originating from within the outer layers of the sarcotesta and penetrating the inner layers of the fertile complex. The surface texture and morphology of the sclerotesta of the seed was also unique to each species. (C) 2004 The Linnean Society of London.

Cultivated Rubus, Ribes and Vaccinium are important, high value crops in many parts of Europe. Other cane and bush fruits e.g. elderberry (Sambucus nigra) and sea buckthorn (Hippophae ramnoides) are locally important. Within the EU, cane and bush fruits are increasing in importance as many countries see this as the way to diversify cropping or gain revenue from exports. Cane and bush fruits have a distinct range of diseases and pests. The severity of attack and levels of damage vary considerably throughout the geographical and climatic zones within Europe. On a global scale, these crops will always be regarded as being 'minor' and as a consequence receive little attention from most major agribusiness companies to develop specific chemicals for diseases and pests control. At present, the range of chemicals permitted varies from country-to-country and until harmonisation is complete, this is likely to remain a problem. The industry is responding to customer demands for low-input production by initiating fundamental changes to the approach to pest and disease control by adopting Integrated Crop Management (ICM) systems. The challenge is to adequately control pests and diseases and still achieve the yield and quality demanded by the consumer. Interactions between delegates have led to a small amount of on-going research, particularly on raspberry beetle (Byturus tomentosus) and the results of a survey on national cane and bush fruit disease and pest research is presented.

Several markers of oxidative processes have been measured in leaves of Phaseolus vulgalis infected with Botrytis cinerea, with the specific objective of investigating changes induced by this necrotrophic pathogen in tissue remote from the lesion. There was a progressive decrease with time in the contents of ascorbic acid (AA) in apparently healthy tissues from infected plants and non-inoculated plants grown under identical high-humidity conditions (abiotically stressed controls), and for periods > 48 h this decrease was greater in the infected plants. This decline in AA content was accompanied by an elevation in the intensity of the electron paramagnetic resonance (EPR) signal from adducts of the spin trap alpha-(4-pyridyl-1-oidde)-N-t-butylnitrone (POBN), a destabilisation of the (monodehydro) ascorbate radical (Asc) signal in the presence of POBN, and an increase in the ratio of Asc to AA in samples studied in the absence of the spin trap. These results are consistent with a shift in redox status to more oxidising conditions in apparently healthy tissue of infected plants and indicate the prevalence of chemical processes that are distinctly different from those in uninfected plants. However, no differences in lipid peroxidation products or the single-peak free radical and Fe(III) (g = 4.27) EPR signals were observed between these tissues distant from the lesions and those from abiotically stressed controls. In addition, the pathogen-derived sesquiterpene toxin botrydial and a second Mn(H) EPR signal, both of which are associated with Botrytis infection, were not detected in these 'apparently healthy' tissues. (C) 2003 Society of Chemical Industry.

Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.

Flying adults are attracted to white traps (Rebell bianco). Monitoring a relationship established between adults caught on traps and subsequent larval damage to ripe fruit. Control threshold proposed for Great Britain and Switzerland <5 beetles per trap for fresh fruit, <20 beetles per trap for processed fruit. The ‘Racer’ Project A Blueprint for Rubus IPM Research S C Gordon1, J A T Woodford1, B Williamson1, A Grassi2, H Hohn3 and T Tuovinen4 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom 2 Isituto Agrario Provinciale di S. Michele all' Adige, Via Edmundo Mach 1, I-38010 S.Michele a/Adige, Italy 3 Eidgenossische Forschungsanstalt fur Obst-, Weinund Gartenbau, Postfach, Schloss, CH-8820 Wadenswil, Switzerland Agricultural Research Centre of Finland, Institute of Plant Protection, FIN-31600 Jokioinen, Finland

Infection of leaves of Arabidopsis thaliana with conidial suspensions of the necrotrophic pathogen Botrytis cinerea resulted in a large decrease in the level of ascorbic acid and increases in intensity of a single-peak free radical and Fe(III) (g=4.27) signals in electron paramagnetic resonance (EPR) spectra. These changes were not confined to the spreading lesions or associated areas of chlorosis, but extended to other apparently healthy tissues in the infected leaves. They are, therefore, consistent with the existence of high levels of oxidative stress being generated as a result of the infection process. The expected accompanying increases in levels of the aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), were not observed, and in the case of MDA the levels in tissue from infected plants were appreciably lower than in the healthy controls. These last findings are surprising and demonstrate a difference in the response of A. thaliana to infection with B. cinerea compared with tissues from other plant families studied previously.

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiaeα-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.

Nuclear magnetic resonance (NMR) imaging was investigated as a tool for the detailed morphological comparison of two species of Podocarpaceae of taxonomic interest; these were difficult to investigate by conventional methods.Two- and three-dimensional NMR images of female cones of Afrocarpus falcatus and Prumnopitys ferruginea were acquired using a range of protocols. Conventional sectioning and microscopic techniques and low-temperature scanning electron microscopy were used where possible, to corroborate the assignments of the NMR images.A three-dimensional network of resin canals in Prumnopitys ferruginea was revealed and the presence of resin confirmed by chemical shift imaging. Similar canals in cones of Afrocarpus falcatus did not contain resin but the presence of spoke-like vascular traces passing through the sclerotesta was demonstrated. marked differences in the structure of the sarcotesta of the two species were readily discernible.NMR imaging allowed noninvasive retrieval of both internal morphological and histochemical information from single specimens in their natural state in a much shorter time than conventional methods would allow and provides useful data for taxonomic purposes in the Podocarpaceae.

A standard quality control test to estimate the rate of decay of harvested fruits of red raspberry (Rubus idaeus) was used to compare the shelf-life of fruits damaged by larvae of raspberry beetle (Byturus tomentosus) and those without visible evidence of larval damage. Samples of fruit of two cultivars, 'Glen Ample' and 'Glen Rosa', were picked from plots managed without insecticides or fungicides at the Scottish Crop Research Institute, and sorted into those with visible larval damage and those that showed no external damage. Harvested fruits were placed individually into separate wells of plastic minipots in sterile seed trays, kept at room temperature (c. 20degrees C) in subdued light and high humidity, and examined at daily intervals for 5 days. Each fruit was discarded when it showed surface mycelium of grey mould, caused by Botrytis cinerea. Records of the number of days until visible rotting occurred were used to estimate the time taken for 10% and 50% of the sample to rot. Fruits damaged by raspberry beetle larvae started to rot more rapidly than undamaged fruits. For both cultivars, 10% of the damaged fruits had started to rot in less than 2 days under the standard conditions, about half a day sooner than had undamaged fruits (P<0.01). Damaged fruits also took less time for 50% of the samples to decay (P<0.001).

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.

Many taxonomic distinctions are made or refined on the basis of herbarium material that is either dried or preserved in spirit medium. Hitherto, examination of internal structure has only been possible by the destructive sectioning of the preserved material. In this paper, the use of nuclear magnetic resonance (NMR) imaging for the non-destructive, non-invasive, complete three-dimensional structural examination of herbarium material is demonstrated for the first time. The experimental materials were the fruiting structures of two species of Southern Hemisphere Podocarpaceae: Acmopyle pancheri and Podocarpus nivalis. Material dried in accordance with standard herbarium techniques was used, as well as material preserved in spirit and freshly gathered fruits. The dried material was subsequently rehydrated using standard techniques, and protocols established for the specimens. Appropriate selection of NMR imaging parameters allowed a variety of anatomical features to be highlighted on a single specimen. Fresh specimens from living material gave the best NMR signals. Dry specimens gave no signal except from the lipid in the seed, but when rehydrated the images yielded almost as much information about internal structure as did a fresh specimen of the same taxon. Thus, NMR imaging has great potential value as a non-invasive method for obtaining details of the internal structure of fruits and seeds and is particularly useful when, as in the case of Acmopyle, the sclerotesta of the seed is too lignified for sectioning by conventional methods.

Free radical adducts of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits ofCapsicum annuum investigated 5 days after infection withBotrytis cinerea.The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.

The developmental morphology and anatomy of the female cones of Acmopyle pancheri (Brongn. & Gris) Pilg. (Podocarpaceae) are described and illustrated, based on observations. histology, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) imaging. Ovulate development is typically podocarpaceous. Ovules are unitegmic, and horizontal or inclined upwards throughout ontogeny: the pollination drop is inverted because of the declinate micropyle. Ontogeny of the epimatium-ovule complex is acropetal, the epimatium developing first. A terminal, distal sterile bract creates a pollen-scavenging area. During development, the whole cone re-orientates through some 270 degrees, and the seed realigns approx. 60 degrees with respect to the receptacle axis. The 'receptacle' or podocarpium supporting the seed is Termed by gradual fusion of initially free bracts. The structures adnate to these bracts represent homologues of ovuliferous scales; they bear vestigial epimatia which may develop into supernumerary ovules or non-functional epimatia. Thus, female cones of A. pancheri are vestigially multi-ovulate. NMR imaging effectively and non-invasively revealed the three-dimensional arrangement of vascular bundles and resin canals in the cones. (C) 2001 Annals of Botany Company.