1Genetic Engineering Laboratory, College of Biotechnology, South China Agricultural University, Guangzhou, China; 2Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, Poland; 3Institute of Plant Breeding and Acclimatization, Strzeszynska 36, 60-479 Poznan, Poland

Background and objectives
Leptosphaeria maculans is a complex species consisting of many forms differing in their host plant range and pathogenicity. The fungal strains isolated from oilseed rape plants with symptoms of blackleg disease are divided, according to their morphological, biochemical and molecular characteristics, into two groups: Tox0 and Tox+. Tox0 isolates do not produce phytotoxins from the sirodesmin group and are regarded to be less pathogenic. This type of isolate prevails in Poland and, as preliminary screening suggests, in the Czech Republic and Slovakia.

Several compounds causing necrotic spots on cotyledons or leaves of different plant species (barley, wheat, pea, lupin, beet, oilseed rape) were found in culture filtrates of different Polish Tox0 isolates. For one of these isolates (PL18) the active compound was identified as benzoic acid [1]. A partially purified phytotoxic fraction from the other isolate (PL68) contained large amounts of succinic acid presumed to be the active principle. The objective of this work was to study the interaction of fungal metabolites with different plant tissues grown in vitro.

Materials and methods
Microspores were isolated from buds of winter oilseed rape DH-0120 line and cultured on NLN medium with 13% of sucrose. Well developed green embryos (about 5 mm long) were placed in 15 ml of NLN medium containing 8% of sucrose in petri dishes with 50 embryos per dish. Petri dishes were placed on a shaker at 50 r.p.m. at 25C with photoperiod 16 h light : 8 h dark.

Protoplasts were isolated from leaves of 2-month-old plants of rape cv. Leo and grown on semi-solid D2a medium with 0.65 M of glucose, NAA, 6-BA and 2,4,5-T. The purified protoplasts were placed in petri dishes at the final density of 3-5x105 per ml. The petri dishes were incubated in the dark without shaking at 26C. Compounds tested were dissolved in media of respective cultures, sterilised using Millex filters and added to final concentrations ranging from 0 to 50 mM. Damaged protoplasts were identified by staining with Evans Blue and observation under an inverse microscope.

Results and conclusions
Sodium benzoate applied to microspore-derived embryos at 16 mM concentration caused 100% of lethal damage after 14 days of treatment. The same effect was observed on protoplasts after 7 days of treatment with 2 mM concentration of this compound. As the treated embryos turned white, disruption of chloroplasts seems to be the possible basis of phytotoxic activity of sodium benzoate. A similar effect was observed after treatment of embryos with a partially purified phytotoxic fraction from isolate PL68. However, the process was much faster in this case. Sodium succinate added to the cultures in concentrations of up to 50 mM did not affect their growth. The overall phytotoxic effect of applied metabolites was much stronger on protoplasts than on intact cells in embryos.

The above results show that in vitro cultures of oilseed rape, including microspore-derived embryos and protoplasts, are useful tools for the bioassay of phytotoxic effects of metabolites from the Tox0 Leptosphaeria maculans isolates.

1. Kachlicki P, Stobiecki M, Jedryczka M, 1996. Rosliny Oleiste 17, 193-198.