Among diseases, fungi are the main cause of yield loss in fruit crops. They are controlled by several traditional techniques including quarantine, sanitation, breeding and clonal selection of resistant varieties and application of fungicides. However, resistant cultivars, with the onset of new strains of virulent pathogens, tend to become susceptible over time. In addition, the unrestrained use of fungicides, as well as increasing production costs and degrading the environment, induce new forms of resistance within pathogens, forcing the development of new pesticides. These problems have encouraged the search for biotechnological solutions to combating fungal disease. At present research is focused on identifying the genes involved in resistance, both those encoding for enzymes involved in the biosynthesis of toxic compounds for fungi and those encoding toxin proteins which directly inhibit fungal growth (Cornelissen and Melchers, 1993; Terras etal., 1998) with the aim of introducing them in susceptible plants orsubstituting their inefficient antifungal gene promoters with more efficient ones. Several proteins have been reported with antifungal activity; they were classified into at least 11 classes named pathogenesis-related proteins (PRs). Some of them also showed antiviral and antibacterial activities.
1. Phenyl propanoid metabolism;
2. Hydrolytic enzymes, such as chitinases and ß-1, 3–glucanases;
3. Hydroxyproline-rich glycoproteins (cell wall proteins);
4. Inhibitors of fungal enzymes, such as PGIP.
Plant ß-1, 3-Glucanases (PR-2) and chitinases (PR-3) represent potential antifungal hydrolases which act synergistically to inhibit fungal growth in vitro (Mauch et al., 1988). In addition, ß-1, 3-Glucanases release glycosidic fragments from both the pathogen and the host cellwalls which could act as signals in the elicitation of host defences (Keen and Yoshikawa, 1983; Hahn et al., 1989; Takeuchi et al., 1990). Many of the genes induced by plant disease- resistance responses encode proteins with direct antifungal activity (AFPs) in vitro (Lamb et al., 1992; Terras et al., 1998). Identification of such anti-fungal proteins was isolated from plants and from the fungus itself such as Tricodermaharsianum (Neuhaus et al., 1991; Mikkelsen et al.,1992; Melchers et al., 1993) and from humans. They include: Defensins, small cysteine-rich peptides, 2S albumins, chitin-binding proteins, lipid-transfer proteins, hydrogen-peroxide-generating enzymes (Terras et al., 1993; Garcia-Olmedo et al., 1998), stilbene synthase (Hain et al., 1990), ribosome inactivating proteins (Stripe et al., 1992; Longemann et al., 1992), lysozyme from humans, osmotin (PR-5) and osmotin-like protein (Liuetal., 1994; Zhu et al., 1996), polygalacturonase-inhibiting protein,thaumatin and several others. Several herbaceous plants have been engineered with some success by using single genes (chitinase, defensin, osmotin, etc.) or multiple genes (osmotin + chitinase + PR1) (Veronese et al., 1999).
Correlation between the level of expression of antifungal proteins in the leaves and resistance has been observed in several herbaceous transgenes. In field trials the olive plants expressing the osmotin gene of tobacco showed reduction of growth (Rugini et al., 2000a; D’Angeli et al., 2001) similarly to the apple plants engineered with the endochitinase gene (Bolar et al., 2000). Research aims at isolating pure compounds (toxins) from fungi, i.e., specific pectic enzymes, malseccin, fusicoccin, fusaric acids, and others to be used as selective pressure on plant cell or tissue culture to recover resistant genotypes, although the resistance acquired by the cells is not always maintained by the derived regenerated plant. However, Orlandoet al. (1997) demonstrated that pectic enzymes of Rhizoctonia fragariae wereeffective in selecting strawberry plants resistant to some fungi. Unfortunately the fruits appeared pale red in colour and a little sour, since horto-di-hydroxiphenols in the leaves were increased by over 40%. In the absence of pure compounds the crude culture filtrate of the pathogen could be applied (Hammerschlag and Ognianov, 1990). In vitro mutation by ionising rays combined with toxin-used selection seems a promising strategy for future work for fruit crops also.
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