ABSTRACT
The fruit and vegetable production industry has undergone major structural changes during recent years owing to new consumer expectations. The pressure to diminish fungicide residues on fruits and vegetables at all points along the supply chain presents even more problems than that of other agricultural products. At present, growers have to conform with regulations that limit undesirable biocide residues while, at the same time, choosing treatments that will maintain the quality of their produce. Losses from postharvest disease can be as high as 25%; they may result from poor handling during harvesting, processing, storage, and/or transportation to the point of sale. In tropical countries losses may be as high as 50% because at elevated temperatures postharvest senescence is accelerated. After harvest, rapid physiological processes are initiated that result in the breakdown of the host resistance mechanism and lead to enhanced development of rots. In fruits, the physiological changes that occur during ripening serve as a signal for initiation of fungal attack and colonization. Despite the magnitude of the problem, the development of new approaches to disease control has not always received priority. This is partly because the abundance of the food supply in developed countries has masked the severity of postharvest losses, but is mostly due to the difficulty of devising treatments that
prolong produce quality while at the same time satisfying consumer demand for reduced biocide residues. The requirement to improve produce quality and to reduce postharvest disease within the limitations imposed by the new marketing controls has stimulated revision of the old techniques and the development of new protocols. These aim at a more holistic approach whereby the chemical control of disease is not the only means considered. Since senescence results in the activation of infections, and improper handling and storage encourages disease development, the new approaches encompass improvements in produce handling and storage, in combination with techniques to enhance host resistance. In the present chapter we will refer firstly to the mechanism of fungal pathogenicity and host-pathogen communication, and secondly to specific cases in which new approaches have resulted in improved quality of the stored produce. MECHANISMS OF HOST SURFACE PENETRATION AND SUBSEQUENT COLONIZATION A number of postharvest pathogens start their disease cycle with a conidium landing on the host surface. The fungus must have evolved strategies to recognize a suitable host, to penetrate and invade plant tissues and to overcome host defences. To perform these tasks, the fun-
gus is capable of perceiving chemical and physical signals from various different host plants and of responding with the appropriate metabolic activities required for pathogenic development. Communication between the fungal conidium and the plant surface begins as soon as the conidium lands on the plant. Some aspects of this interaction are specific to the host whereas others are relatively nonspecific, and depend only on the lipophilic nature of the plant cuticle. Fungal conidia are often covered with a lipophilic self-inhibitor when they arrive on the plant surface. Diffusion of the self-inhibitor into the hydrophobic plant cuticle relieves the self-inhibition and allows germination of the conidia. This concept was demonstrated with the conidia of Magnaporthe grisea (Hegde and Kolattukudy, 1997): the conidial surface material was recovered by washing with organic solvents and was found to inhibit conidial germination in a dosedependent manner; this inhibition was reversed by plant-surface wax. Colletotrichum gloeosporioides self-inhibitors, although they have not been identified, are known to be lipophilic (Tsurushima et al., 1995) and the selfinhibition is probably relieved by diffusion of the inhibitor into the host cuticle. How self-inhibitors prevent conidial germination is not known, but they would be expected to cause suppression of early gene expression (Chitarra et al., 2005). Since the cam (calmodulin) gene of M. grisea was found to be expressed very early during the conidial interaction with the host (Liu and Kolattukudy, 1999), it was chosen as a test gene to examine the effects of self-inhibitors. Cam gene promoter-driven expression of green fluorescent protein (GFP) reporter gene in M. grisea was inhibited by self-inhibitors whose effect was reversed when the self-inhibition was relieved by the addition of plant-surface wax. Surface attachment was required for cam promoterdriven GFP expression and appressorium formation, and both of these were inhibited by concanavalin that inhibits conidial surface attachment. Beside the self-inhibitory conidial factors, it has been known for some time that fungal conidia require contact with a hard surface
before they can be induced to germinate and to differentiate into appressoria. The molecular basis of this requirement has not been elucidated. A differential display approach was used to identify some of the fungal genes of C. gloeosporioides that are induced by contact with a hard surface (chip genes). One such gene was identified as that which encodes a 16.2-kDa ubiquitin-conjugating enzyme; this gene complemented the ubc5 yeast mutant (Liu and Kolattukudy, 1998). Thus, one role of contact with a hard surface is to induce ubiquitindependent protein degradation, which is involved in conidial germination and appressorial differentiation. Two other Colletotrichum hard-surface-induced protein genes (chip genes) that were discovered by differential display encode CHIP2 and CHIP3, two novel proteins of 65 and 64 kDa, respectively. CHIP2 contains a putative nuclear localization signal, a leucine zipper motif and a heptad repeat region which might dimerize into a coiled-coil structure. The targets of this putative transcription factor and its biological function are unknown. CHIP3 contains nine transmembrane domains. Although induction of Chip2 and Chip3 by hard surface contact was confirmed, their biological functions remain unknown (Kim et al., 2000a). Also, Ca+2 calmodulin signaling is probably activated by contact of the conidia with a hard surface (Kim et al., 1998). The C. gloeosporioides calmodulin gene (cam) showed almost 90% identity with other fungal cam genes. The 1.3kb cam transcript level was elevated more than tenfold by contact of the conidia with a hard surface for one hour and, furthermore, a calmodulin antagonist severely inhibited germination and appressorium formation (Kim et al., 1998). Thus, the cam gene product seems to be involved in the induction of conidial germination and appressorial differentiation. Involvement of calmodulin signaling in germination and appressorium formation would involve calmodulin kinase (CaMK). camK transcript was also obtained from a cDNA library prepared from hard-surface-induced transcripts isolated from C. gloeosporioides conidia. The identity of the camK gene was confirmed by demonstrating CaMK activity of the cloned
gene product expressed in E. coli (Kolattukudy et al., 2000). Involvement of CaMK in germination and appressorium formation was strongly suggested by the finding that the CaMK selective inhibitor, KN93, inhibited phosphorylation of proteins that were found to be associated with hard-surface treatment of the fungal conidia. Kim et al., (2002) also reported another novel gene (Chip 6) that was induced by contact of C. gloeosporioides with a hard surface; it encodes a sterol glycosyl transferase, as confirmed by the measurement of glycosyl transferase activity of the gene product expressed in E. coli. This glycosyl transferase was identified as a novel pathogenesis gene, since its disruption caused a drastic decrease in the virulence, although the mutants grew normally and formed normal-looking appressoria (Kim et al., 2002). This suggests that conidia of postharvest pathogens sense and react to various stimuli on the fruit, even before penetration. In light of the importance of volatiles produced by fruits, it will be of interest to search for their effect on the initial stages of pathogenicity. The biotrophic stage After invading the host, fungi use various strategies to gain access to host nutrients. Whereas necrotrophs quickly kill plant cells in order to feed subsequently as saprotrophs, other fungi maintain biotrophic relationships with their hosts either transiently or until sporulation. Most of the postharvest pathogens are considered to be necrotrophs, e.g., Botrytis cinerea, Alternaria alternata, Penicillium spp. The biotrophic lifestyle is realized in a remarkable range of ways: intercellular (Cladosporium fulvum); subcuticular (Venturia inaequalis); interand intracellular (Claviceps purpurea, Ustilago maydis, monokaryotic rust fungi); extracellular with haustoria within epidermal cells (powdery mildews); intercellular with haustoria within parenchyma cells (dikaryotic rust fungi and downy mildews). A transient type of biotrophy followed by necrotrophy is observed in the so-called hemibiotrophic fungi (M. grisea, Phytophthora infestans and Colletotrichum spp.) (Mendgen and Hahn, 2001). These are regarded as the hemibiotrophic fungi members
of the genus Colletotrichum initially grow within the cell walls of host epidermal cells leading to the formation of long-term biotrophic or quiescent infections. After penetration, the intracellular infection vesicle and the primary hyphae colonize only a few host cells, and both are surrounded by a matrix that separates the fungal cell wall from the invaginated host plasma membrane (Mendgen and Hahn, 2001). This matrix is extracytoplasmic and is connected to the plant apoplast. It seems that the existence of a matrix layer is crucial for the biotrophic life style. Within the interfacial matrix, a fungal glycoprotein, encoded by CIH1, was identified. The protein was shown to be present uniquely at this interface in the biotrophic stage of hemibiotrophic Colletotrichum spp.; its expression was switched off at the onset of necrotrophic development. The completion of the biotrophic stage, which is a quiescent stage, might be the result of a host signal accompanied by a signal transduction process that leads to the initiation of processes leading to the destruction of the plant cell. Induction of penetrating structures The first host barrier to be breached is the cuticle, which covers all aerial parts of the plant. The cuticle consists of cutin, an insoluble polyester composed mainly of two families of hydroxy and hydroxy-epoxy fatty acids, derived from the most common cell fatty acids: one derived from C16 fatty acids and the other from C18 unsaturated fatty acids. The monomers of some plants are mainly of the C16 family, whereas others are mixtures of the C16 and C18 families. There is a layer (associated with the cutin layer) consisting of complex mixture of soluble lipids, collectively called waxes, that are distinctly different from cell lipids. The most common major components of cuticular waxes are hydrocarbons and their oxygenated derivatives such as secondary alcohols and ketones, very long-chain fatty acids, aldehydes and alcohols, and wax esters composed of very-long-chain fatty alcohols and fatty acids. Very-long-chain β-diketones and pentacyclic triterpenes are sometimes major components of waxes, particularly on stems and fruits of some plants. As if to provide chemical and metabolic
stability, the cuticle-associated waxes, which function at the surface exposed to oxygen and other atmosphere components as well as to microbes, are mostly saturated and are usually hard for microbes to absorb and metabolize (Kolattukudy, 1996). If the signals at the plant surface are perceived as favorable by the fungi, conidia germinate and the germ tube differentiates into an infection structure called an appressorium, which produces the infection peg that penetrates into the host. In other fungi where no signals are perceived or appressorium are not produced, their germ tubes penetrate the cuticle directly or through wounds.
