H. G. Azinheira; M. C. Silva; L. Guerra Guimarães, K. Mendgen**, C. Rodrigues Jr. ; C. Pinto Ricardo*
Centro de Investigação das Ferrugens do Cafeeiro (IICT). Quinta do Marquês 2784-505 Oeiras, *Instituto de Tecnologia Química Biológica, Rua da Quinta Grande, 6, 2784-505 Oeriras. Portugal.
**Universität Konstanz, Fakultät für Biologie, Konstanz, Germany
Summary. Rust fungi differentiate a series of complex infection structures in order to successfully infect their hosts. The most striking difficulty to study obligate biotrophic fungi are the absolute need of host cell. Although the use of thigmotropically inductive membranes allowed the differentiation of infection structures in somerust, the ability of uredospores of the orange coffee rust to develop infection structures in the absence of its host plant is not known. In this work we evaluated a variety of membranes such as scratched polyethylene, coated membranes and collodion membranes supplemented with mineral oil as well as membranes sprayed with concanavalin A, boiled driselase and cell walls to induce differentiation inHemileia vastatrix Berk. & Br. The development of infection structures was followed by light microscopy. On all the membranes assayed uredospores formed germination tubes and appressoria.
Keywords: Rust; Differentiation; Artificial membranes; Infection structures; Thigmodifferentiation.
Infection by uredospores of rust fungi is preceded by development of specializedinfection structures required for host penetration. The precise signal provided by the stoma to induce the fungal differentiation is not know. Dickinson (1949,1971) was the first to use artificial membranes to induce the development of infection structures. After his work, an increasing number of evidence has suggested that some physical characteristics of the stomatal structures are responsible forstopping polarized growth of the germ tube leading to appressorium formation. During appressorium development the cytoplasm migrates into the swelling germ tube tip. The developmental process is accompanied by many notable cytological changes including mitosis of both nuclei and the formation of a septum that ultimately isolates the apressorium from the cytoplasmic emptied germ tube (Lamboy et al.,1995). The mechanism by which fungal cells detect topographical signals is not well understood. The signalling process must connect the thigmosensing receptor system at the exterior with the cell interior where signal mediation culminates in various cytological, biochemical and morphological changes
Although a great diversity of rusts has been tested aiming the production of infection structuresin vitro no work was performed with H. vastatrix so far. Here we report experiments with this rust to check its ability to differentiate in vitro and compare the nuclear condition of uredospores, germlings and appressoria with that observed in vivo. The formation of infection structures was followed by light microscopy.
Material and methods:
Leaves of Coffea arabica L. or membranes wereinoculated with fresh uredospores of H. vastatrix as described by D’ Oliveira & Rodrigues (1961).
Collodion membranes were prepared as described by Heath & Heath (1976).
Cell walls (CW) preparations were obtained from 3 grams of coffee leaves (healthy and infected) by grinding in liquid nitrogen and suspending in 250 mL bidistilled water. To remove debris the leaf homogenate was passed throughcheesecloth. The filtrate was stirred for 30 minutes and centrifuged (676 g, 15 min. at 4ºC). The pellet was ressuspended in 250 mL of water kept stirred for 30 minutes and then centrifuged. The pellet was washed again and centrifuged (9670 g, 4 min. at 4ºC). The final pellet was suspended in agar (1.5%) and gelatine (1%) previously boiled. This suspension was sprayed on polyethylene and collodion...