A study of some biochemical and histopathological responses of wet-stored recalcitrant seeds of Avicennia marina infected by Fusarium moniliforme

Abstract

Although fungi cause a recognized problem during storage of recalcitrant seeds of many tropical species, there are no data to date on defence strategies of these seeds against fungal attack. To ascertain whether recalcitrant seeds of Avicennia marina elaborate compounds that might suppress fungal proliferation during hydrated storage, the production and efficacy of beta-1,3-glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) were studied in relation to histopathological changes. Freshly harvested seeds had low beta-1,3-glucanase and chitinase activities and fluorescence microscopy revealed progressive deterioration of the internal tissues of these seeds associated with fungal infection during hydrated storage. In seeds treated to minimize associated fungi (clean seeds), beta-1,3-glucanase and chitinase activities increased significantly during 10 d of hydrated storage. Similar high levels of activity were observed when these seeds were experimentally infected with Fusarium moniliforme and subjected to further storage. The histopathological observations indicated delayed disease development in the 10-d clean-storage period, although the hypersensitive response was not observed. The results suggest that, although the recalcitrant seeds of A. marina elaborate some antifungal enzymes, there is a lack of effective defence strategies that might lead to successful responses against fungal infections.

Fig. 1. Epifluorescence micrographs of the distal hypocotyl tip of excised embryonic axes taken from (A–C) freshly harvested seeds (0c0i); (D–F) seeds stored clean for 4 d (4c0i); (G–I) seeds stored clean for 10 d (10c0i). In A–C, the bright blue fluorescence reveals chitin and hence fungal structures. Although the occasional bright-light-blue fluorescing chitin, indicating a fungal structure, could be seen on the bristles (A) there was no evidence of fungal infection on the hypocotyl surface (B) or within the tissue of a root primordium (C). A and C, ×100; B, ×40. In D–F, no signs of fungal infection could be seen on the bristles (D), the surface of the hypocotyl tip (E), or internal tissues (F). D and E, ×40; F, ×100. In G–I, fungal structures revealed by the bright blue fluorescence of chitin could be observed on the bristles (G) and the surface of the root primordium (H). Fungal penetration into the root primordium tissues (I) was mainly confined to the epidermal cells. G and I, ×100; H, ×40. * indicates a root primordium.

Fig. 2. Epifluorescence micrographs of the distal hypocotyl tip of excised embryonic axes taken from (A–C) seeds inoculated when fresh and then stored for 8 d (0c8i); (D–F) seeds stored clean for 4 d, inoculated and then stored for 8 d (4c8i); (G–I) seeds stored clean for 10 d, inoculated and then stored for 8 d (10c8i). In A–C, most of the fungal mass, revealed by bright blue fluorescence of chitin, was confined to the bristles (A) and the adjacent surfaces of the hypocotyls tip (B); however, localized but substantial regions of the internal tissue were colonized by fungus (C), and degradation of the internal tissue was also apparent. A–C, ×100. In D–F, although some fungal mycelium, revealed by the bright blue fluorescence of chitin, can be seen on the bristles (D), the hypocotyl surface (E) and internal tissue was clean (F). D, 100×; E and F, 40×. In G–I, fungal mycelium, revealed by the bright blue fluorescence of chitin, was common on the bristles (G), on and below the hypocotyl surface (H), and the occasional hypha (arrow) could also be discerned in the internal tissue (I). G and H, ×100; I, ×200.

Fig. 3. Changes in the total β-1,3-glucanase activity in the axes of recalcitrant seeds of Avicenia marina. Data presented are the means of three replicates. Error bars represent positive s.d. Data from the first experiment only are presented, but similar trends were obtained in confirmatory studies. 0c0i, Fresh seeds; 4c0i, seeds stored clean for 4 d, 10c0i, seeds stored clean for 10 d; 0c8i, seeds stored infected for 8 d; 4c8i; seeds stored clean for 4 d, infected and re-stored for 8 d; 10c8i, seeds stored clean for 10 d, infected and re-stored for 8 d.

Fig. 4. Changes in the total chitinase activity in axes of recalcitrant seeds of Avicenia marina. Data presented are the means of three replicates. Error bars represent positive s.d. Data from first experiment only are presented as similar trends were obtained in confirmatory studies.