5.2.18
PHYTOTOXIC METABOLITES PRODUCED BY ASCOCHYTA CAULINA, A PROMISING BIOCONTROL AGENT OF CHENOPODIUM ALBUM
M VURRO1, A EVIDENTE2, R CAPASSO2, A ANDOLFI2, O TAGLIALATELA-SCAFATI3, MC ZONNO1 and A MOTTA4

1Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, I-70125 Bari, Italy; 2Dipartimento di Scienze Chimico-Agrarie, Universitą di Napoli Federico II, I-80055 Portici, Italy; 3Dipartimento di Chimica delle Sostanze Naturali, Universitą di Napoli Federico II, I-80131 Napoli, Italy; 4Istituto per la Chimica di Molecole di Interesse Biologico, CNR, I-80072 Arco Felice, Italy

Background and objectives
Chenopodium album, also known as common lambsquarter, is an important weed widely distributed in arable crops. In Europe it is considered to be the most important weed in 10 major crops, due to the abundance, competitiveness and longevity of seeds in soil [1]. Because it has developed resistance to some herbicides, C. album is considered a good target for biological control. The use of a fungal pathogen, Ascochyta caulina, to control this noxious weed has recently been proposed [2]. In growth-chamber experiments, application of pycnidiospores of the fungus to C. album plants caused the appearance of large necrosis on leaves and stems and, depending on the amount of necrosis developed, plants showed retarded growth or death. The genus Ascochyta comprises a large number of species that are pathogenic to plants. Several Ascochyta species elaborate a number of phytotoxic compounds which could be of importance in the plant disease they cause [3]. Considering the kind of symptoms caused by A. caulina and the possible use of fungal toxins as an alternative or in addition to the use of pathogens in weed biocontrol [4], it is of interest to investigate the production of toxic metabolites by A. caulina, and carry out their isolation, as well as their chemical and biological characterization.

Results and conclusions
The culture filtrate obtained growing the fungus in a defined liquid medium, which showed high phytotoxicity on leaves and cuttings of both host and non-host plants, was examined to ascertain the chemical nature of the phytotoxic metabolites. They proved to be hydrophilic substances, remaining in the aqueous phase after exhaustive extraction of the culture filtrate with organic solvents of increased polarity. As deduced by dialysis experiments, phytotoxic metabolites had a molecular weight lower than 1000 Da. These results prompted the purification of the culture filtrate by gel filtration using a Biol-Gel P-2 column, eluted with ultrapure water; 14 groups of homogeneous fractions were obtained, and only groups 4, 6 and 7 showed phytotoxic activity. The residue obtained by group 4 proved to be the only homogeneous compound (235 mg/l), as shown by TLC analysis on silica gel and reverse phase. This was named ascaulitoxin. The application of droplets (15 µl) containing the toxin (30 µg) on punctured-detached host leaves caused the appearance of wide necrosis similar to that caused by the pathogen. Ascaulitoxin also caused toxic effects on cuttings and on germinating seeds. However, no antibiotic and zootoxic effects were observed at the concentrations used. The chemical and biological characterization of ascaulitoxin, as well as of other phytotoxic metabolites present in the filtrate, will be described in this communication. The possible direct or indirect use of pure metabolites or partially purified filtrates in the biological control programmes will be discussed.

References
1. Schroeder D, Mueller-Scharer H, Stinson CS, 1993. Weed Research 33, 449-458.
2. Kempenaar C, 1995. Thesis, Wageningen, The Netherlands.
3. Strange RN, 1997. In Upadhyay RK, Mukerji KG, eds, Toxins in Plant Disease Development and Evolving Biotechnology. Oxford & IBH Publishing, pp. 167-181.
4. Strobel GA, Kenfield D, Bunkers G et al., 1991. Experientia 47, 819-826.