ABSTRACT

Production of chitinases is an operable mechanism in antagonism of bacteria and fungi. Serratia plymuthica C48 was isolated from the rhizosphere of oilseed rape antagonistic to phytopathogenic fungi. S. plymuthica C48 was shown to produce and excrete a set of various chitinases. The chitinolytic system consists of two endochitinases, a chitobiosidase and, at least two N-acetyl-ß-1,4-D-hexosaminidases including a chitobiase. For the endochitinase with an apparent molecular mass of 65 kDa a high homology to chitinase A from Serratia marcescens by comparison of N-terminal amino acid sequences was discovered. S. plymuthica C48 significantly suppress the growth of the fungus Verticillium longisporum while a chemically constructed chitinase-deficient mutant C48/3Rif ^r chi^- did not exhibit antifungal activity. A decrease of the percentage of diseased plants by root application of wildtype bacteria in greenhouse was shown supporting the important role of cell wall degrading enzymes for antifungal activities.

INTRODUCTION

Chitin, an insoluble linear 1,4-ß-glucosidically linked polymer of N-acetylglucosamine (GlcNac), is a major structural component of most fungal cell walls. Several chitinolytic bacteria and fungi, e. g Serratia marcescens, Aeromonas caviae, Enterobacter agglomerans and Trichoderma harzianum have been shown to be potent biological control agents (BCA’s) protecting plants against pathogenic fungi (Ordentlich 1988; Tronsmo 1991; Inbar and Chet 1991; Chernin 1995).

Verticillium longisporum (Stark) Karapapa et al. 1997; syn. Verticillium dahliae var. longisporum is an important pathogen of oilseed rape (Brassica napus L.) causing tracheomycosis and wilts. Microsclerotia of the fungus are able to germinate and infect plant roots all over the vegetation period. They may outlast more than 14 years in soil. Therefore, the use of chemical fungicides is insufficient and, the application of BCA’s seems to be promising (Fravel 1988). S. plymuthica C48 is a natural occurring rhizobacterium of oilseed rape with chitinolytic activity (Kalbe et al. 1996).

In this study the chitinolytic system of S. plymuthica C48, the involvement of chitinases in antagonism with fungal plant pathogens and, the efficiency of biocontrol in oilseed rape was investigated.

Material and Methods

Cultures and growth media. The strain C48 was isolated from the rhizosphere of oilseed rape (Brassica napus L.; Kalbe et al. 1996). Bacteria were grown at 30 °C in nutrient broth (Sifin, Berlin, Germany), in liquid semiminimal medium [(SM): 1.62 g nutrient broth, 0.5 g NaCl, 6 g M9 salts, 15 g bacto-agar (Difco Laboratories, Detroit, USA), 1 l distilled water with 0.1 mM CaCl₂, 1 mM MgSO₄, 3 nM Thiamin-HCl (all from Sigma Chemical Co., St. Louis, USA)] supplemented with 0.2 % colloidal chitin (Hirano and Nagao 1988) or 0.2 % glucose. S. plymuthica C48 (DSMZ 12502, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and V. longisporum V25 (host plant: oilseed rape) were from the culture collection of the Institute of Microbiology, University of Rostock.

Detection of chitinolytic activity on plates. Bacterial colonies were screened for chitinase production and secretion by plating on SM agar with 0.2 % colloidal chitin as the sole carbon source.

Mutagenesis. Mutagenesis of S. plymuthica C48 was done by treatment with N-methyl-N'-nitro-N-nitroso-guanidine (NTG) (Sigma) as described previously (Frankowski et al. 1998). One mutant C48/3Rif ^r chi^- was selected for further studies.

Fungal inhibition assay. The ability of the bacteria to produce antifungal substances against V. longisporum V25 was determined by the paired in vitro assay on Waksman agar (Berg 1996).

Preparation of extracellular and proteins. Cells were grown in 50 ml SM with 0.2 % colloidal chitin or 0.2 % glucose for up to 120 h at 30 °C. The cells were centrifuged and, the supernatant was filtered (0.22 µm; Sartorius, Göttingen, Germany). For analysis by gel electrophoresis the samples were concentrated at 4 °C in Ultrafiltration cell (Amicon, Beverly, USA) using a 10 kDa molecular weight cutoff membrane.

Assay of chitinolytic activity in solutions. The enzymatic activity by using assays with chromogenic p-nitrophenole (pNP) analogs of di-, tri- and tetrasaccharids of N-acetyl-ß-D-glucosamine (all from Sigma) according to the method of Roberts and Selitrennikoff (1988) was measured. The release of the chromophore pNP from the substrates was measured at 410 nm. The U of enzymatic activity was defined as 1 µmol of released pNP per minute. Protein concentrations by the method of Bradford (1976) were determined.

Detection of chitinolytic enzymes after gel electrophoresis. Concentrated proteins were separated by 4 % native or 12 % sodium dodecyl sulfate (SDS) polyacryl-amide gel electrophoresis (PAGE) (Laemmli 1970). Native PAGE was done as described by Frankowski et al. (1998). Chitinases were visible after overlaying the gel with agarose substrate solution and incubation for 60 min at 40 °C in a moist chamber (Tronsmo and Harman 1993). The molecular weights of chitinases were estimated by using SDS-PAGE broad range standards (Sigma).

Greenhouse and field trials. Soil samples (compost, peat, field soil, 1:1:2, vol.) with V. longisporum (10 g microsclerotia per kg soil) were infested. Polypropylene boxes (7x7x8 cm) with infested soil and planted rape seeds cv. Lambada were filled. Bacteria were suspended in 0.85 % NaCl solution and applicated at 1.E+08 cfu g^-1 fw. The plants were grown at 20-30 °C with daily irrigation. Two field trials on natural infested field soil with random disposition of coated rape seeds were done from August 1997 to July 1998 and from April to June 1998. For seed coating a mixture of bacteria, nutrient broth, agar agar and gum arabic dissolved in 0.85 % NaCl solution was used. The disease index according to Knaape (1994) was determined.

Results

Antifungal activity. S. plymuthica C48 was found to supress the growth of Verticillium longisporum V25 in vitro with inhibition zones of about 10 mm after 5 days of incubation on Waksman agar at 22 °C. In contrast, the mutant C48/3 Rif ^r chi^- was not able to inhibit the growing of the phytopathogenic fungus.

Chitinolytic activity. S. plymuthica C48 was able to hydrolyze colloidal chitin. Large clearing zones caused by the hydrolysis of colloidal chitin were obtained after 72 to 96 h of growth (SM agar, 0.2 % chitin) at 30 °C. Mutant C48/3 Rif ^r chi^- did not form any clearing zone on plates even after longer time of cultivation. Chitinase production after cultivation in liquid culture (SM, 0.2 % colloidal chitin) was determined. A high level of chitinolytic activity in extracellular proteins at day 3 of cultivation and remained stable in culture supernatant until day 5. All of the tested chitin analoga could be hydrolyzed. When chitin in the medium was replaced with glucose (0.2 %) the strain C48 showed no detectable extracellular chitinase activity. Mutant C48/3 Rif ^r chi^- did not show extracellular chitinolytic activity after cultivation on chitin or glucose.

Detection of chitinolytic enzymes. Extracellular chitinolytic enzymes were detected using a set of three fluorescent chitin derivatives. After separation with native PAGE three different activity bands in culture supernatant of S. plymuthica C48 were detected, and the proteins were named CHITA, CHITB and CHITC, respectively. Activity of the enzyme CHITA only with MUF-GlcNac was detected while CHITB activity with all three substrates could be determined. Activity of CHITC only with the analogon of triacetylchitotriose [MUF-(GlcNac)₂] was observed. After SDS-PAGE four distinct enzymes, designated according to their apparent molecular masses as CHIT100, CHIT65, CHIT54 and CHIT37, were detected in the same sample. CHIT100 and CHIT54 activities were discovered by using all of the three substrates, and CHIT65 and CHIT37 activities with analogs of chitotriose [MUF-(GlcNac)₂] and chitotetraose [MUF-(GlcNac)₃] were detected. After SDS-PAGE and Coomassie protein staining the N-terminal amino acid sequence of the corresponding protein band of CHIT65 was determined to be ATPGKPTLAWGNTKFAI.

Biocontrol activity. In greenhouse a significant reduction of diseased plants in the variant with the wildtype strain of C48 was obtained. While the disease indices of the untreated control and the chitinase deficient mutant reached 21 % the index in the test with S. plymuthica C48 wildtype was decreased to 7 %. At the end of greenhouse trial the bacteria in abundances of 5.E+05 cfu g^-1 fw were counted.

In the variants with applicated wildtype in both field trials non significant increases of yields by 3 % (1997/98) and 1.4 % (1998) respectively were determined. The abundances of applicated bacteria were 2.E+05 cfu g^-1 fw (1997/98) on average.

Discussion

In 1994 the strain S. plymuthica C48 was isolated from the rhizosphere of oilseed rape and characterized as an antagonist to phytopathogenic fungi such as Verticillium dahliae, Rhizoctonia solani and Sclerotinia sclerotiorum and as a potential biological control agent (Kalbe et al. 1996). The strain was found to produce siderophores and antibiotic-like substances (our unpublished observation). Direct antifungal effect may be based on antibiosis and production of lytic enzymes (Chet et al. 1990). S. plymuthica C48 exhibited strong chitinolytic activity as determined by degradation of colloidal chitin in liquid medium and on agar plates. Additionally, a high activity by the release of pNP and MUF from chitooligosaccharide analoga was estimated. A set of three fluorescent chitin derivatives was used to identify the chitinolytic activity in extracellular proteins renatured following their separation by SDS-PAGE or by native PAGE. According to the nomenclature suggested by Sahai and Manocha (1993) the strain S. plymuthica C48 produced all three types of chitinases. The ability for synthesis of a chitinase system by other bacteria like Serratia marcescens and Enterobacter agglomerans (Chernin et al. 1995, Tews et al. 1996, Watanabe et al. 1997) was described. There is a wealth of data supporting the important role of chitinolytic enzymes in antifungal activities of various antagonistic microorganisms (Ordentlich et al. 1988, Shapira et al. 1989, Inbar and Chet 1991, Chernin et al. 1997, Frankowski et al. 1998).

Chemical induced mutant C48/3 Rif ^r chi^- deficient in chitinolytic activity was used to validate the importance of the chitinolytic system for antifungal activity. As shown in this study this mutant is unable to synthetize and excrete any chitinolytic enzyme resulting in no antifungal effect in vitro. The lack of biocontrol activity of this mutant in comparison to S. plymuthica C48 wildtype in greenhouse was shown supporting the importance of cell wall degrading enzymes for biocontrol applications. However, in field the wildtype strain due to the very low abundances was not able to protect the plants and to prevent yield losses significantly. Studies for suitable application and formulation methods of bacteria are in progress.

CONCLUSION

S. plymuthica C 48 is an important antagonist and biological control agent against Verticillium wilt in oilseed rape. The results presented in this work confirm the role of the chitinolytic system in the antifungal activity. For successful commercialization more knowledge about the formulation of the BCA is necessary.

ACKNOWLEDGEMENTS

The authors thank Robert Schmid (University of Osnabrück) for cooperation and determination of the N-terminal amino acid sequences and Hella Goschke for valuable technical assistance.

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