Microscopical observations of Sphaerellopsis filum, a parasite of Puccinia recondita

Sphaerellopsis filum is a well-known parasite associated with many species of rust fungi. It is of frequent occurrence as parasite of cereal rusts: Puccinia recondita, P. coronata, P. graminis, P. hordei and P. striiformis. Uredial sori of Puccinia recondita f.sp. tritici infected with Sphaerellopsis filum were examined by light and scanning microscope to determine morphology of hyperparasite as well as the parasite-hyperparasite contact. The microscopical examination of infected uredinia clearly showed the intimate connection of S. filum with its rust host.


INTRODUCTION
Sphaerellopsis filum (Biv.ex Fr.) B.Sutton [Darluca filum (Biv.ex Fr.) Castagne], anamorph of Eudarluca caricis (Fr.)O. Erikss., is a cosmopolitan hyperparasite associated with 369 rust species belonging to 30 genera.It has been found in more than 50 countries (K r a n z and B r a n d e n b u r g e r, 1981).It has been mostly reported to penetrate uredial sori, also occasionally the telial and ecial sori as well.Sphaerellopsis filum is most commonly observed as anamorph producing clumps of shiny, black spherical pycnidia situated among the spores of uredial sori, where it is presumed to derive nutrients from direct hyphal penetration of uredospores.Because of this penetration, the uredospores number is reduced and spore production in some cases completely stopped.Due to this fact the hyperparasite can potentially be an important biocontrolling factor (Yu a n et al., 1998 This study clarifies the hyperparasitic relationship between Sphaerellopsis filum and Puccinia recondita Rob.ex Desm.f. sp.tritici (Erikss.)Johnson, describes the nutrient-securing structures and demonstrates the elements of morphology of hyperparasite.

MATERIAL AND METHODS
Samples of wheat infected with Puccinia recondita and Sphaerellopsis filum were collected in September 2002 from an experimental field of Department of Plant Pathology in Warsaw.The uredial sori free of S. filum infection has been used as control.Light microscopy observations were carried out on a fresh rust infected leaf fragment.Pycnidia were removed from uredial sori, placed on a microscope slide in a drop of distilled water and covered with a cover glass.Dimensions of pycnidia and conidia were measured by examining 10 pycnidia and 50 spores flowing out of a squashed pycnidia.
For scanning electron microscopy small, dry leaf pieces (1 sq.cm diameter) with and without hyperparasite colonies were excised, afterwards thinly coated with carbon and silver in an JEOL JEE-4C vacuum evaporator and finally examined with a JEOL JSM-35 scanning electron microscope operating at 25 kV.The microscopical observations were carried out in the Laboratory of Electron Microscopy of the Warsaw Agricultural University.
The scanning microscope observations show that uredial sori were often completely overgrown with mycelium of S. filum.White and loose hyphae of hyperparasite were developed on the pustule surface and inside it (fig.4).Sphaerellopsis filum was intimately associated with Puccinia recondita uredospores penetrating spores by specialized structures such as appressoria (fig.5).Many of the uredospores in contact with a hyperparasite collapsed, disintegrated and were occasionally divested of spikes on the surface of spore walls (fig.6).

DISCUSSION
Microscope observations of infected uredial sori clearly demonstrated the intimate association of Sphaerellopsis filum and Puccinia recondita.
B a r n e t t and B i n d e r (1973) classified mycoparasites into two groups.The necrotrophic parasites, which kill the host cells before or just after invasion were assigned to the first group.The dead cells are the nutrients for these parasites.The second group are biotrophs which are able to obtain nutrients from live host cells.These parasites may derive the nutrients forming absorbtive cells of specialized hyphae haustoria and hyphal swellings appressoria.
The previous ultrastructural examination of Sphaerellopsis filum (C a r l i n g et al., 1976) demonstrated that S. filum is a biotroph penetrating and causing the destruction of uredospores of Puccinia graminis.Carling affirmed that this penetration may be due to combined mechanical and enzymatic processes, with absence of specialized penetration structures.
This study confirms that Sphaerellopsis filum is a biotrophic mycoparasite, whose hyphae parasites on living uredospores, causing their progressive destruction.The specialized hyphae of hyperparasite form swelled structures similar to appressoria adhesing to the surface of uredospores (figs. 5, 6).These penetration structures haven't been previously observed.
Observation concerning the morphology of pycnidia and conidia of S. filum confirms their previous descriptions (C a l p o u z o s et al., 1957al., , Yu a n et al., 1998)).