3.5.1
MOLECULAR ANALYSIS OF BIOCONTROL OF VERTICILLIUM WILT BY CHITINOLYTIC SERRATIA PLYMUTHICA C48
MOLECULAR ANALYSIS OF BIOCONTROL OF VERTICILLIUM WILT BY CHITINOLYTIC SERRATIA PLYMUTHICA C48

JENS FRANKOWSKI, HUBERT BAHL and GABRIELE BERG

University of Rostock, Microbiology, Gertrudenstr. 11a, D-18051 Rostock, Germany

Background and Objectives
Serratia plymuthica is a common rhizobacterium with antifungal effects to phytopathogenic fungi, e.g., the soil-borne fungus Verticillium longisporum causing tracheomycosis and wilts in oilseed rape. The mechanisms responsible for antifungal activity, are obviously due to the production of antibiotics, lytic enzymes and siderophores. The antifungal mechanisms were specific for each of 16 investigated strains [1]. The objectives of the investigation was to characterize the chitinolytic system of Serratia plymuthica C48 and the importance of chitinases in biological control of Verticillium wilt.

Materials and Methods
The strain of Serratia plymuthica was isolated from the rhizosphere of oilseed rape [1]. The chitinolytic enzymes were characterized by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivates of N-acetylglucoseamine. Additionally, fluorescent substrates with a 4-methylumbelliferyl group linked by b-1,4 linkage to N-acetylglucoseamin mono- or oligosaccharide were used to identify the chitinolytic activity of proteins after gel electrophoresis [2]. Mutants without chitinolytic activity were obtained after treatment with N-methyl-N’-nitro-N-nitrosoguanidine and Tn5-mutagenesis. Wildtype and mutants were tested for biological control of Verticillium wilt in a greenhouse and in naturally infested fields.

Results and Conclusions
Four enzymes of Serratia plymuthica C48 with chitinolytic activity were detected: two endochitinases (E.C.3.2.1.14) of 73 and 108 kDa and two ß-N-acetylglucosaminidases (E.C.3.2.52) of 82 and 130 and kDa. The N-terminal amino acid sequence of the 108 kDa-endochitinase is similar to the chitinase A of Serratia marcescens (provided by R. Schmid, Osnabrück, Germany). Mutants without chitinolytic activity were obtained after treatmant with N-methyl-N'-nitro-N-nitrosoguanidine. While the wild-type C48 shows inhibition zones up to 20 mm in dual culture against V. longisporum, no inhibition was found for a part of mutants. Therefore, we conclude that the chitinolytic activity is exclusively responsible for the antifungal activity of Serratia plymuthica C48 in vitro. The mutant S. plymuthica C48chi-3/4 was unable to protect plants of oilseed rape against Verticillium wilt in the greenhouse. Additionally, a collection of Kanamycin-resistant transconjugants generated by random Tn5 insertions into the genome of S. plymuthica were screened for loss of antifungal activity in vitro. Some inactive mutants were deficient in parts of the chitinolytic system and are ideal to study the function of different chitinolytic enzymes in biocontrol of plant pathogens.

References
1. Kalbe K, Marten P, Berg, G, 1996. Microbiol. Research 151, 4433-4400.
2. Chernin L, Ismailov Z, Haran S, Chet I, 1995. Applied Environmental Microbiology 61, 1720-1726.