In vitro sensitivity and tolerance of Fusarium solani towards chitinases and [beta]-1,3-glucanases

In agriculture, fungal diseases have always been one of the major problems. Many options exist to combat the pathogens responsible. Application of fungicides is for specific diseases a very effective means of control. However, new strains of fungal pathogens may emerge showing resistance to such compounds. Moreover, environmental and health concerns have made these chemicals less favourable. Crop rotation is a possibility to control disease, but is economically less attractive for farmers. Traditional plant breeding to obtain resistant cultivars fits best in a system of sustainable agriculture. However, this technology is very laborious and time consuming. Also, desired resistance traits might not be available within the species or even within related species. Since the development of technology for genetic engineering of plants, new strategies for introducing resistance in plants to fungal pathogens have emerged.In the first chapter of this thesis, a review is presented on the various strategies that are used or could possibly be used in the future to genetically engineer fungal resistance. One of the strategies followed at MOGEN involves overexpression of one or more antifungal proteins. The work, presented in this thesis, is part of this strategy. An in vitro assay had been established to assist in the isolation and identification of such antifungal proteins (Woloshuk et al., 1991) and has played a pivotal role in the results described here. In search for such antifungal proteins, the phenomenon of induced resistance is exploited. Nicotiana tabacum, cv. Samsun NN, when inoculated with tobacco mosaic virus (TMV), acquires resistance to subsequent pathogen attack. Synthesis of a large number of pathogenesis-related (PR) proteins is induced (Linthorst, 1991).In Chapter 2 results are described using protein extracts from tobacco leaves inoculated with TMV. These induced extracts were calibrated for the levels of known PR-proteins and tested in vitro on a variety of fungi. The majority of fungi were inhibited in growth by these extracts. Spores of all fungi were far more sensitive to induced protein extracts if pregerminated before addition of the extracts, when compared to assaying without pregermination.The natural location of many antifungal tobacco PR-proteins, such as Chi-I, Glu-I and AP24, is the vacuole. However, since many pathogens reside in the intercellular spaces, overexpression of these proteins is expected not to yield the desired protective effect. Therefore, genes were modified in such a way that proteins, in stead of being targeted to the vacuole, were rerouted extracellularly. Results of these experiments are presented in Chapter 3.In Chapters 4 and 5 several of the tobacco PR-proteins were purified and assayed for their in vitro antifungal effects. In Chapter 4, the proteins of group PR-2, β-1,3-glucanases, and PR-3, chitinases, were assayed for their antifungal activity, either alone or in synergy. Apoplastic 5, the isolation, enzymatic activity and antifungal activity of the class I PR-4 CBP20, is described.In Chapter 6, the proteins from transgenic plants described in Chapter 4, were reisolated in order to analyze whether extracellular targeting had affected antifungal activity.As observed in Chapter 2, non pregerminated fungal spores were far less sensitive to induced protein extracts compared to germlings. In Chapters 7 and 8, the phenomenon of decreased sensitivity occurring during incubation with antifungal proteins is further investigated using F.solani f.sp. phaseoli as a model system. The effect of gerniination time before addition of proteins was studied. Results presented indicate that macroconidia adapt to the presence of specific chitinases only during the first three hours of germination. Concomitantly, as described in Chapter 8, specific protease(s) are released by the germinating spore capable of cleaving the chitin-binding domain from Chi-I, and CBP20. The influence of this chitin-binding domain on the level of antifungal activity of Chi-I, and CBP20 as well as its role on the adaptation phenomenon has been determined.The overall results described in this thesis are summarized in Chapter 9. The use of an in vitro assay to assist in the isolation of antifungal proteins is addressed in detail. Since it was demonstrated that macroconidia can adapt to the presence of specific antifungal proteins, the relevance of this observation is discussed. Finally, the importance of in vitro identification of antifungal proteins in engineering fungal resistant plants is demonstrated