5.2.69
PRODUCTION OF EXTRACELLULAR ENZYMES BY ANTAGONISTIC FUNGI OF FUSARIUM SPECIES CAUSING FOOT ROT OF WHEAT

R ROBERTI1, E ZAKRISSON2, A BRUNELLI1 and A CESARI1

1DIPROVAL, Plant Protection Division, University of Bologna, Via Filippo Re 8, 40126 Bologna, Italy; 2Sveriges Lantbruksuniversitet, Box 44, 23053 Alnarp, Sweden

Background and objectives
Foot rot, principally caused by species of Fusarium, is a common disease of wheat in Italy. The disease may be seed- and soil-transmitted and can reduce stands and yield of surviving plants. On the basis of results of field and greenhouse trials carried out with antagonistic fungi applied on wheat seeds [1], five selected antagonistic fungi (Trichoderma koningii 1A, T. viride 144, T. harzianum 312, Gliocladium roseum 11A and Penicillium frequentans) were tested for their ability to produce the extracellular enzymes N-acetyl-P-D-glucosaminidase and chitin 1,4-O-chitobiosidase.

Materials and methods
Antagonistic fungi were grown with cell-wall components of the pathogenic fungi Fusarium culmorum, F. graminearum, F. nivale or F. avenaceum as sole carbon sources. To quantitate the enzymes, the release of p-nitrophenol from p-nitrophenyl-N-acetyl-P-D-glucosaminide [2] or p-nitrophenyl-P-N,N'-diacetyl chitobiose [3] was assayed. The antagonists were also tested for the production of non-volatile substances released in the medium, using the Dennis and Webster technique [4].

Results and conclusions
All the tested antagonistic fungi produced both of the extracellular enzymes to a various extent. The activity of N-acetyl-P-D-glucosaminidase was generally higher than that of chitin 1,4-p-chitobiosidase. G. roseum 11A produced the highest amount of both enzymes and this was very evident in the presence of F. nivale cell walls; the second highest level of chitinolitic activity was secreted by T. viride 144 in the case of N-acetyl-P-D-glucosaminidase and by T. viride 144 or T. harzianum 312 when regarding 1,4-p-chitobiosidase. Similarly all the antagonists released non-volatile substances in the medium, able to reduce pathogen growth: T. harzianum 312 completely inhibited the growth of all the pathogens; T. viride 144 and T. koningii 1A were also good producers of non-volatile substances active in reducing colony growth, whereas G. roseum, even though able to produce high levels of chitinolitic enzymes, reduced the pathogen growth less effectively.

We can conclude that chitinolytic activities from the tested antagonists may play a role in digesting cell walls of foot root pathogens despite the different enzyme levels released in the medium. On the basis of the presumptive various modes of action of the antagonists, further experiments are also in progress to explain the different effectiveness of G. roseum in producing extracellular enzymes and inhibiting pathogen growth.

References
1. Roberti R, Flori P, Brunelli A, 1995. Proceedings of a conference on Microbial Control Agents in Sustainable Agriculture, Saint Vincent, p. 200.
2. Roberts WK, Selitrennikoff CP, 1988. Journal of General Microbiology 134, 169-176.
3. Ohatara A, 1988. In Wood WA, Kellogg ST, eds, Methods of Enzymology (Academic Press, pp. 462-470.
4. Dennis C, Webster J, 1971. Transactions of the British Mycological Society 57, 25-48.