Sulphur Deficiency Causes a Reduction in Antimicrobial Potential and Leads to Increased Disease Susceptibility of Oilseed Rape

The reduction of atmospheric sulphur dioxide pollution is causing increasing problems of sulphur deficiency in sulphur-demanding crop plants in northern Europe. Elemental sulphur and many sulphur containing compounds such as cysteine-rich antifungal proteins, glucosinolates (GSL) and phytoalexins play important roles in plant disease resistance. The aim of this work was to analyse the effect of inadequate sulphur supply on disease resistance of oilseed rape (Brassica napus). Compared with fertilized oilseed rape, healthy looking S-deficient plants showed increased susceptibility to the blackleg fungus Leptosphaeria maculans, to the generalist necrotroph Botrytis cinerea and to the oomycete Phytophthora brassicae. To analyse possible causes of the increased disease susceptibility of S-deficient plants, protein extracts and methanolic extracts of secondary metabolites of plants grown with and without adequate sulphur supply were tested for antimicrobial activity. None of the protein extracts showed antimicrobial activity. However, extracts containing secondary metabolites from normally grown plants showed a strong antimicrobial activity in in vitro tests with various fungal and bacterial pathogens. This activity was almost totally lost in extracts derived from S-deficient plants. The antimicrobial activity did not appear to be based on the activity of phytoalexins because it was present in healthy plants and was not increased by a previous inoculation with Botrytis cinerea. The loss of antifungal activity in S-deficient plants correlated with a strong reduction of various GSL, thus suggesting a reduced level of GSL as a possible cause of the reduced antimicrobial potential. However, limited tests of commercially available GSL or their degradation products did not demonstrate a causal link. Our results show that S-deficiency of oilseed rape negatively affects disease resistance and suggest that this effect is at least partially caused by a reduction of sulphur-dependent phytoanticipins. Introduction Sulphur is an essential macroelement for plant life and has numerous biological functions (Leustek et al., 2000). It is taken up by plants in its inorganic sulphate form from the soil or as sulphur dioxide and hydrogen sulphide from the atmosphere. Plants assimilate and reduce sulphate to sulphide which is incorporated into cysteine and further converted to methionine. Sulphurcontaining compounds play crucial roles in a number of cellular processes, such as redox reactions, detoxification of heavy metals and xenobiotics, and metabolism of secondary products (Saito, 2000; Nikiforova et al., 2003). Intensive farming has greatly increased the demand for sulphur in crop production in the last decades. This development was largely unnoticed in industrialized countries because of concomitant pollution by atmospheric deposition of sulphur dioxide produced by the burning of S-containing fossil fuels. In the second part of the 20th century air pollution with sulphur dioxide became a major concern (http://www.ourplanet. com). In response to this global problem governments started to enforce a drastic reduction of sulphur dioxide emissions (Helsinki Protocol, 1979). As an unexpected outcome of the successful reduction of S-pollution an increased frequency of sulphur deficiency was to be observed in several crops mainly in northern Europe. Inadequate sulphur supply was becoming a factor limiting crop yield and quality (Dämmgen et al., 1998; Eriksen and Mortensen, 1999). It has been known since antiquity that sulphur has protective effects against pests and diseases. Most of the knowledge is however restricted to the external effect of foliar applied inorganic sulphur. Less is known about soil supplied sulphur which has a strong influence on U. S. Copyright Clearance Centre Code Statement: 0931–1785/2005/1531–0027 $ 15.00/0 www.blackwell-synergy.com J. Phytopathology 153, 27–36 (2005) 2005 Blackwell Verlag, Berlin ISSN 0931-1785 plant resistance by directly stimulating biochemical processes in primary and secondary metabolism (Pezet et al., 1986; Schnug, 1996). Field observations pointed to a positive correlation between S-fertilization and enhanced disease resistance against fungal pathogens, leading to the question, whether sulphate availability could be a limiting factor for the ability of plants to fight off pathogens (Davidson and Goss, 1972; Schnug, 1996). Many compounds that play a role in active defence against pathogens contain sulphur including cysteine-rich antifungal peptides, phytoalexins and glucosinolates (GSL) (Hell, 1997; Pedras et al., 2000). In addition, elemental sulphur (S) has been described as a component of disease resistance in certain plant species (Williams and Cooper, 2003, 2004). In recent years oilseed rape (Brassica napus L.) has become a major crop in Europe and one of the major oil crops worldwide (Graner et al., 2003). Because of its high demand for sulphur, oilseed rape is particularly sensitive to S-deficiency. It produces seeds with a high content of sulphur rich proteins (Zhao et al., 1997; Blake-Kalff et al., 1998) and requires sulphur for the synthesis of GSL, a group of thioglucoside compounds (Blake-Kalff et al., 1998), and S-containing phytoalexins (Hammerschmidt and Dann, 1999). We have analysed the effect of inadequate S-supply on the resistance of oilseed rape to different pathogens. Of the phytopathogenic fungi known to affect oilseed rape, the ascomycete fungus Leptosphaeria maculans (anamorph: Phoma lingam) causes the highest economic losses worldwide (Howlett et al., 2001). Leptosphaeria maculans is a specific pathogen of Brassica species and is a facultative necrotroph initially growing biotrophically and later promoting necrosis to live saprophytically on dead plant material (Hammond et al., 1985; Hammond and Lewis, 1987). Phytophthora brassicae is an hemibiotrophic oomycete which is able to infect a wide range of Crucifer species including B. napus and Arabidopsis thaliana (Man in’t Veld et al., 2002; Si-Ammour et al., 2003). Botrytis cinerea (teleomorph Botryotinia fuckeliana), commonly named grey mould, is a necrotrophic pathogen with a broad host range (MacFarlane, 1968). It was chosen as a nonspecialized ubiquitous pathogen for comparison with the specialized L. maculans. Our results show a clear effect of the S-nutritional status of the plant on its disease resistance against all three pathogens. The reduced disease resistance of S-deficient plants correlated with a strong reduction of antimicrobial activity of plant extracts and a greatly reduced content of various GSL. Materials and Methods Organisms and growth conditions Seeds of Brassica napus cv. Bienvenu (0) and cv. Express (00) were obtained from the Swiss Federal Agricultural Research Station of Plant Production in Changins. Seeds were sown on vermiculite and watered first with tap water and after 10 days once with half strength Hoagland solution containing 0.5 mm MgSO4 (Hoagland and Martin, 1950). After 15 days the seedlings were transferred to pots (diameter 12 cm) containing quartz sand and further watered with 0.5· Hoagland solution with or without addition of 0.5 mm MgSO4. For the S-deficient plants the MgSO4 was replaced by equimolar amount of MgCl2, and among micronutrients CuCl2, MnCl2 and ZnCl2 were used instead of CuSO4, MnSO4 and ZnSO4. Plants were grown in a growth chamber with 20 C, 16 h light/ 18 C, 8 h dark cycle. Plants were used for experiments 5–6 weeks after planting which corresponded to a period of 3–4 weeks of sulphur starvation. Leptosphaeria maculans (anamorph: Phoma lingam), Penicillium digitatum and Cladosporium sp. isolates were obtained from the Swiss Federal Agricultural Research Station of Plant Production in Changins and grown on potato dextrose agar (PDA; Difco, Detroit, MI, USA) containing 25 lg/ml of aureomycin (chlortetracycline hydrochloride; Rectolab SA, Servion, Switzerland). Leptosphaeria maculans pycnidiospore production was induced by growing colonies for 14 days at 15 C under a 12 h black light (OSRAM L3673 BLB)/12 h dark cycle. Pycnidiospores were harvested according to Hammond et al. (1985). The Botrytis cinerea isolates BMM (Zimmerli et al., 2001) and Pellier were isolated respectively from geranium and vine and grown on PDA. Conidia from 14-day old well-sporulating colonies were harvested in distilled water containing 0.2% (v/v) Tween 20. The oomycete P. brassicae constitutively expressing a green fluorescent protein (GFP) was grown on V8 agar (Si-Ammour et al., 2003). Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000) was grown in Luria broth with 25 lg/ml rifampicin. Inoculation protocols Leaves of 5–6-week-old plants, at growth stage 2.4–2.5 of the scale according to Harper and Berkenkamp (1975), were inoculated with the different pathogens. For Leptosphaeria maculans 10 ll of a spore suspension (10 spores/ml) in water containing 0.2% (v/v) Tween 20 was applied to the leaves after wounding with a needle. Plants were incubated for 5 days in 100% relative humidity in a glass chamber before the lids were removed. Lesion size was measured at 12 and/or 21 dpi. For Botrytis cinerea 10 ll of a spore suspension (10 spores/ml) in water containing 0.2% (v/v) Tween 20 was applied to the leaves after wounding with a needle. Plants were incubated at 100% relative humidity and lesion size was measured at 4 or 5 dpi. Agar plugs taken from the margins of an expanding colony of P. brassicae isolate HH (SiAmmour et al., 2003) were applied to the leaves after wounding with a needle. Plants were then incubated in 100% relative humidity for 7 days for lesion size measurement. GFP fluorescence was determined 4 days after the inoculation (Si-Ammour et al., 2003). Measurements of total sulphur and glutathione For total S content, leaves of 6-week-old B. napus cv. Bienvenu plants were dried for 48 h at 65 C and then 28 Dubuis et al.