ABSTRACT

Experiments were designed based on the hypothesis that phytopathogenic fungi of Brassica can overcome the plant glucosinolates-myrosinase defense system by degradation or biotransformation of glucosinolates to non-toxic or less toxic products. Isolates of Phoma lingam, Verticillium dahliae and Sclerotinia sclerotiorum were tested for their degradation ability of glucosinolates in vitro. The results showed that the isolates of Phoma lingam and Verticillium dahliae could efficiently degrade the aliphatic glucosinolates sinigrin, progoitrin and gluconapin as well as the aromatic glucosinolate sinalbin. The degradation had no inhibitory effects on the spore germination and mycelial growth rate, which suggested that the degradation is a detoxifying process. Sclerotinia sclerotiorum could not degrade any of these glucosinolates, indicating that this fungal pathogen employed another way to overcome glucosinolates-myrosinase system. Both virulent and avirulent isolates of Phoma lingam could degrade glucosinolates indicated that the glucosinolates degrading ability of fungal pathogens is a necessary factor other than a determinant in pathogenesis.

INTRODUCTION

In vitro toxicity tests had proved that some glucosinolate products are toxic or inhibitory to many fungal pathogens of Brassica and non-Brassica species. For example, 2-propenyl isothiocyanate derived from the glucosinolates sinigrin was very toxic to Leptosphaeria maculans ( Mithen and Lewis, 1986, Peterka and Schlosser, 1989 ). However, many phytopathogenic fungi, including Leptosphaeria maculans, are still able to infect glucosinolate containing plants. This phenomenon imply that fungal pathogens of Brassica have developed certain mechanism to overcome glucosinolate-myrosinase defense system, but little is known about fungal resistance to glucosinolate breakdown products (Osbourn, 1996). The compartmentalisation of glucosinolates and myrosinases in different plant tissue and cells suggest that there are at least three possible ways for fungal pathogens to overcome glucosinolate-myrosinase defense system of their hosts. Firstly, the pathogen can degrade or transform intact glucosinolate to non-toxic or less toxic products before they are degraded to toxic products by myrosinases endogenous to plant; Secondly, pathogens may have developed certain mechanism to detoxify toxic glucosinolates degradation products, such as cyanide hydratase described in Leptosphaeria maculans, which can detoxify nitriles ( Sexton and Howlett, 1998); Thirdly, pathogens could release certain products during the pathogenesis , which could inhibit the hydrolyses of glucosinolates catalyzed by myrosinase. The first alternative for the pathogens to combat the glucosinolate-myrosinase system is advantageous since it would provent any exposure of toxic products to the invader and at the same time also provide glucose as a nutritional source. There have been data present earlier that microorganisms indeed are capable of metabolizing glucosinolates (Ohtsuru, et al., 1969, Tani, et al., 1974, Rabot, et al., 1995 ), but whether phytopathogenic fungi of Brassica can break down glucosinolates is not known.

In this study, experiments were designed to study whether intact glucosinolates could be degraded by phytopathogenic fungi of Brassica..

MATERIALS AND METHODS

The glucosinolate sinigrin was purchased from Sigma , progoitrin, gluconapin and sinalbin were isolated and purified from rapeseed tissue by solvent extraction followed by RP-HPLC according to a slightly modified protocal from Bjorkqvist ( 1988 ). The identity and purity of the glucosinolates were confirmed by GC-MS analysis. An isolate of Sclerotinia sclerotiorum collected in China, an isolate of Verticilliumdahliae from Svalof Weibull AB and a virulent isolate PHW 1245 and an avirulent isolate PHW1220 of Phoma lingam were used in the experiment. Spore suspensions or mycelial agar disks (4mm diameter ) were inoculated in potato liquid media containing different concentration of glucosinolate. The glucosinolate content in the media was analysed by RP-HPLC and used as an index for evaluation of the glucosinolates degradation capiability of fungal pathogens. Myrosinase activity was also assayed using a glucose oxidase kit from Boehringer.

RESULTS

1. In vitro degradation of sinigrin by phytopathogenic fungi of Brassica

An In vitro test demonstrated that the capability of sinigrin degradation varies among different fungal pathogens ( fig.1 ). Isolates of P. lingam and V. dahliae efficiently degrade sinigrin, while no degradation was found in the case of S.sclerotiorum. The hydrolysis of sinigrin catalyzed by V. dahliae was obviously more rapid compared to P. lingam . The virulent isolate PHW1245 and avirulent PHW1220 of P. lingam have similar sinigrin degradation capability.

2.Degradation of progoitrin, gluconapin and sinalbin by phytopathogenic fungi of Brassica

After inoculation of liquid media with fungal pathogens, the aliphatic glucosinolate progoitrin and gluconapin were completely consumed by isolates of P. lingam and V. dahliae after 10 days. The aromatic glucosinolate sinalbin was also completely degraded by the two isolates of P. lingam while 82.6% of sinabin in the liquid culture containing V. dahliae disappeared at the selected conditions. The isolate of S.sclerotiorum did not have the capability to metabolize any of the glucosinolates tested.

3.The effects of sinigrin degradation on the fungal growth

Mycelial radial growth assay indicated that the hydrolysis of sinigrin catalyzed by fungal pathogens had no significant effects on the fungal radial growth rate. However, when dry weight measurements of fungi from liquid culture experiment were used to assess the effects of degradation, increasing levels of sinigrin in the liquid culture without adding D-glucose were found to be beneficial to the mycelial growth of the virulent isolate PHW 1245 of P. lingam ( fig 2 ), HPLC analysis indicated that all levels of sinigrin added were completely degraded or consumed. But increasing sinigrin levels in the liquid culture added 2% D-glucose had no significant effects on fungal growth, sinigrin content assay showed that no degradation was found in the liquid media with 2% D-glucose .

4 Purification of glucosinolate detoxifying enzymes produced by phytopathogenic fungi

Glucosinolate detoxifying enzymes was partially purified through Ammonium salfate precipitation and ion exchange on a FPLC instrument. A thioglucosidase activity of the crude enzyme fraction was detected by measuring the liberation of glucose when the crude enzymes and sinigrin were mixed.

Western blot analysis using the monoclonal antibody 3D7, which is specific for the plant endogenous myrosinase, revealed a cross-reaction with the fungal sample.

Discussion

Isolates of P. lingam and V. dahliae have the capability to degrade certain glucosinolates. The degradation was not inhibitory to the fungi suggesting that fungal pathogens may overcome glucosinolate-myrosinase defense system by degradation or transformation of intact glucosinolate to non-toxic or less toxic products to prevent the release of toxic glucosinolate degradation products catalyzed by myrosinase endogenous to the plantas. S.sclerotiorum may employ another strategy to circumvent glucosinolate-myrosinase system.

Since both virulent and avirulent isolates of Phoma lingam could degrade glucosinolates this indicates that the glucosinolate degrading ability of fungal pathogens is a necessary factor other than a determinant in pathogenesis.

The results suggest that the attempts to improve the resistance oilseed rape, and other Brassica species, to fungal pathogens by modifying the glucosinolate profile must consider which glucosinolates that can be detoxified by fungal pathogens for a successful outcome.

ACKNOWLEDGEMENTS

The authors thank the Swedish Council for Forestry and Agricultural Research and The Swedish Foundation for Strategic Research or support of the studies, and China Scholarship Council for providing scholarship.

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