Infection Strategies of Botrytis cinerea

Botrytis cinerea is a ubiquitous filamentous fungal pathogen of a wide range of plant species. The fungus is able to infect all aerial parts of its host plants to a certain extent. Infection may cause enormous damage both during plant growth and in the post-harvest phase (during cold storage or transport). B. cinerea is a major cause of economic loss in the production chain of cut flowers, bulb flowers and pot plants. Molecular-genetic studies performed over the past decade have provided a wealth of novel insights into the infection mechanisms utilised by the pathogen. Fungal genes were identified that are important for successful infection by B. cinerea. Such knowledge provides perspectives for designing novel, rational plant protection strategies that effectively counteract important fungal virulence factors. In this review I will divide the infection process into different stages and discuss the role of various fungal enzymes and metabolites in the individual stages. Finally some perspectives are addressed for novel control strategies that may reduce and/or delay the damage inflicted by B. cinerea infection. INTRODUCTION Botrytis cinerea Persoon: Fries (teleomorph Botryotinia fuckeliana, also known as “grey mould fungus”) causes serious preand post-harvest diseases in at least 200 plant species (Jarvis, 1977), including agronomically important crops and harvested commodities, such as grapevine, tomato, strawberry, cucumber, bulb flowers, cut flowers and ornamental plants. The pathogen is a necrotroph, inducing host cell death resulting in serious damage to plant tissues culminating in rot of the plant or the harvested product. Reviews have been published on the infection mechanisms of B. cinerea, with emphasis on microscopic and biochemical studies (Staples and Mayer, 1995) or molecular genetics (Prins et al., 2000b). The application of molecular-genetic tools such as transformation (Hamada et al., 1994), differential gene expression analysis (Benito et al., 1996), gene cloning (van der Vlugt-Bergmans et al., 1997a,b) and targeted mutagenesis (van Kan et al., 1997) has led to novel insight in the structure of B. cinerea genes and their role in the infection process. Here our current understanding of the mechanisms that B. cinerea exploits to infect its host plants will be summarised. DISEASE CYCLE For the purpose of simplification, different stages are distinguished in the disease cycle of B. cinerea (Fig. 1). The disease cycle starts with a conidium landing on the host surface. B. cinerea conidia are ubiquitous in the air and can be transported by wind over long distances before infecting the next host (Jarvis, 1977). Following attachment, the conidium germinates on the host surface under moist conditions and produces a germ tube that develops into an appressorium that penetrates the host surface. The underlying cells are killed and the fungus establishes a primary lesion, in which necrosis and host defense responses may occur. In some cases this is the onset of a period of quiescence of an undefined length, in which fungal outgrowth is restricted (reviewed by Prusky, 1996). Quiescence is especially an important feature in host species infected at the flowering stage (e.g. strawberry). At a certain stage, the defense barriers are breached and the fungus starts a vigorous outgrowth, resulting in rapid maceration of plant tissue, on which the fungus eventually sporulates to produce inoculum for the next infection. Under Proc. VIII IS Postharvest Phys. Ornamentals Eds. N. Marissen et al. Acta Hort. 669, ISHS 2005 78 optimal conditions, an infection cycle may be completed in as little as 3-4 days, depending on the type of host tissue attacked. In the following paragraphs I will discuss the different stages in the disease cycle and the role that enzymes and metabolites (mainly of fungal origin) play in these stages. Given the wide host range of the fungus, not all stages or processes may occur in every infection. Attachment of Conidia Attachment is thought to be mediated by physical surface interactions on the plant cuticle. Two steps were distinguished in the attachment to host tissue. The first stage, preceding the hydration of conidia, typically involves weak adhesive forces resulting from hydrophobic interactions between host and conidial surfaces (Doss et al., 1993). Stronger binding occurs in the second stage (Doss et al., 1995), several hours after inoculation, when conidia have germinated. The tips of germ tubes are covered with fibrillar-like extracellular matrix material (Doss et al., 1995; Cole et al., 1996; see also Fig. 2 in Prins et al., 2000b), consisting of carbohydrates and proteins (Doss, 1999). The matrix contains fungal enzymes (Gil-Ad et al., 2001). It may act as an adhesive on the host surface (Doss et al. 1995) and protect hyphae from dehydration and defense mechanisms of the host. Germination Several factors influence the germination of a conidium. Free surface water or high relative humidity (>93% RH) is essential to germinate and penetrate the host epidermis (Williamson et al., 1995). When dry conidia are inoculated on plant surfaces and incubated in the absence of free surface water, the emerging germ tube usually remains short before it penetrates the surface (Salinas and Verhoeff, 1995; Williamson et al., 1995; Cole et al., 1996). Inoculation with conidia in an aqueous suspension requires the addition of nutrients (Harper et al., 1981; van den Heuvel, 1981), which might mimic the situation in a wound on the plant epidermis, from which nutrients leach. A highly efficient germination and synchronous infection of tomato leaves was obtained, when conidia were pre-incubated for 2-4 hours in liquid medium supplemented with phosphate and sugar, prior to inoculation (Benito et al., 1998). Gaseous compounds may stimulate germination. Elad and Volpin (1988) found a correlation between the level of ethylene production by rose cultivars and the severity of grey mould symptoms. Stimulation of grey mould development by ethylene was also found in strawberry, tomato, cucumber and pepper (e.g. Elad and Volpin, 1988; McNicol et al., 1989). This observation is usually ascribed to host tissue senescence that coincides with ethylene production. The influence of ethylene on B. cinerea itself has not been studied in detail. Analogous to Colletotrichum gloeosporioides (Flaishman and Kolattukudy, 1994), germination of B. cinerea seems to be influenced by ethylene. Germination of conidia on a hydrophobic surface was stimulated by exogenous ethylene, but the germ tube length was unaffected (Kepczynski and Kepczynska, 1977). Application of 2,5-norbornadiene, a competitive inhibitor of ethylene perception in plants, inhibited germination in a reversible manner (Kepczynska, 1993). In a hydrophilic environment, however, ethylene stimulated germ tube elongation without affecting the percentage of germination (Barkai-Golan et al., 1989). Ethylene produced by the plant during tissue senescence or fruit ripening might function as a signal for the conidia on the (hydrophobic) plant surface to germinate and initiate the infection. Germ tube elongation might subsequently be stimulated by ethylene in the more hydrophilic environment of the invaded plant tissue. Thus, ethylene might favour grey mould development by weakening the host, as well as by stimulating germination of B. cinerea conidia and outgrowth of hyphae. Molecular and biochemical approaches are required to further elucidate the effects of ethylene on B. cinerea. Differentiation of Infection Structures on the Host Surface B. cinerea forms appressoria during penetration, albeit not the highly organized