Research on the development of Mycosphaerella graminicola ( Fuckel ) Schroeter teleomorph on wheat leaves from Poland and Czech Republic

Summa r y The aim of the work was to study the development of pseudothecia of M. graminicola. The experiment was carried out in 2002/2003. Wheat leaves collected at Koñczewice (near Toruñ), S3upia Wielka (near Poznañ) and Kromìøí• (Czech Republic) were kept on an experimental field in Warsaw. The pseudothecia were observed under the light and transmission electron microscopy. The first pseudothecia were observed on the dead leaves at the end of July. The largest number of fruiting bodies were noted in October. During the whole period of observation fruiting bodies contained asci in several stages of development. Most of them were immature. In spring the pseudothecia were not found.


INTRODUCTION
Septoria tritici blotch caused by Mycosphaerella graminicola (Fuckel) Schroeter (anamorph: Septoria tritici Rob.ex Desm.) is one of the main wheat leaf necrotic spot diseases.The pathogen infects mainly Triticum aestivum L. and Triticum durum L. and occasionally X Triticosecale Witt., Secale cereale L. and some grass species (We b e r, 1922; Wi l l i a m s and J o n e s , 1973; B r o k e n s h i r e , 1975; Z a m o rs k i and S c h o l l e n b e r g e r, 1995; Z a m o r s k i et al., 1996b).
Under favourable conditions the yield losses in wheat culture range from 30 up to 50% (K e m a , 1996).The intensity of the disease depends on the cultivar susceptibility, the availability of inoculum, crop management and predisposing environmental Until the end of 70th M. graminicola and Septoria tritici were considered to be the separate species.As late as in 1972 Sanderson connected the perfect stage M. graminicola with imperfect stage S. tritici (S a n d e r s o n , 1972, 1976).
So far the sexual stage (teleomorph) of M. graminicola has been identified in many countries of the world, namely Australia (B r o w n et al.,1978)

MATERIALS AND METHODS
The experiment was carried out in 2002/2003.Wheat leaves collected at Koñczewice (near Toruñ), S³upia Wielka (near Poznañ) and Kromìøí• (Czech Republic) were kept on the experimental field in Warsaw.To determine seasonal occurrence of pseudothecia and the stage of their development samples of the leaves (from Koñczewice and S³upia Wielka) were taken once a week in summer and autumn and twice a month in winter and spring.A hundred of pseudothecia, asci and ascospores have been measured to determine their size.The pseudothecia were investigated under the light (Olympus BX 50 ) and transmission electron microscope (JEOL type JEM 1220) in the Laboratory of Electron Microscopy of the Warsaw Agricultural University.
Leaves bearing fruiting bodies were cut into 5 x 2 mm pieces and fixed for 2 h in 4% paraformaldehyde and 5% glutaralaldehyde (pH 7,2-7,3).Material was rinsed in 0,2 M cacodylic buffer (4 x 15 min.)and post-fixed with 2% buffered osmium tetraoxide for 2 h.Materials were dehydrated in ethanol and acetone series and embedded in resin.Polymerisation lasted 24 h.at 60 o C. Ultra-thin sections were cut with Diatome diamond knife by the Reichert Ultracut E Microtome, mounted on copper grids, stained for 20 min. in uranyl acetate and Reynold's citrate for 30 min.Thick sections for light microscope observations were cut by the same ultramicrotome but with glass knife and stained with methylene blue 2% and azure B 1%.
On the leaves from Kromìøí• occurrence of pseudothecia and their development were observed using light microscope only.

RESULTS
The first pseudothecia with developed asci of M. graminicola were observed on the dead leaves at the end of July.The largest number of fruiting bodies were noted in October.
During the whole period of observation pseudothecia contained asci in several stages of development (asci with granulated cytoplasm without ascospores, asci with immature ascospores, asci with mature ascospores) were noted (Fig. 1-4).Most of asci was immature.The biggest percentage of asci in pseudothecia with mature ascospores was observed in September (average 18%) and October (average 22%) (Fig 5).The courses of air temperatures and rainfalls are represented Fig. 6.
The empty pseudothecia, after total ascospores release were observed as well.In spring the pseudothecia on the winter wheat leaves were not found.
The development of pseudothecia of M. graminicola on the leaves from the Czech Republic was similar.al., 1999) the biggest number of pseudothecia with mature ascospores was observed in autumn (September, October), whereas in New Zealand in spring (April, May).This phenomenon can be probably explained by different weather conditions.
In France H a l a m a (1996) observed the fruiting bodies mainly on the leaves.However in United Kingdom pseudothecia were found on leaf sheaths more readily than on leaf blades possibly because the leaf sheaths decompose later than leaves blades (S c o t t et al., 1988).In Poland the pseudothecia were observed on leaves like in France.
conditions (S i m o n and C o r d o , 1998).Heavily infected leaves die prematurely (E y a l et al., 1987; Z a m o r s k i and N o w i c k i , 1997).

Fig. 1 .
Fig. 1.The first phase of asci development Fig. 2. Pseudothecium with immature asci with granulated cytoplasm Because the ascospores can be carried by the wind for many kilometres (S a nd e r s o n and H a m p t o n , 1978) it is suggested that they can be the efficient source of primary infection.
(B r o w n et al., 1978; S a n d e r s o n and H a m p t o n , 1978; E y a l et al., 1987; S c o t t et al., 1988; Ve r r e t , 1995; H a l a m a , 1996; S h a w, 1999).The occurrence of teleomorph in Poland was reported in1996 (Z a m o r s k i et al., 1996a) but there is a lack of information concerning the development of this stage in our country.