Applied Microbial and Cell Physiology

Sclerotinia sclerotiorum causes serious yield losses in oilseed crops worldwide. Bacillus subtilis Tu-100 significantly reduced (P≤0.05) the incidence of disease caused by S. sclerotiorum on oilseed rape at harvest in two trials conducted in fields artificially infested with this pathogen. Mean plant dry weight was significantly greater (P≤0.05) and mean plant length was significantly greater (P≤0.07) at the seven-true-leaf stage with the Tu-100 treatment than with the control. Mean seed yield per 120 plants at harvest was significantly greater (P≤0.05) in the second field trial with treatments containing isolate Tu-100. B. subtilis Tu-100 also promoted the growth of hydroponically grown oilseed rape. Plants were approximately 15% greater in dry weight (P≤0.0001) and 6% greater in length (P≤0.0025) when grown in the presence of isolate Tu-100 in Hoagland’s solution, compared with the noninoculated control. In gnotobiotic studies, the lacZ-tagged strain B. subtilis Tu-100(pUC18) was detected within all roots of oilseed rape. Isolate Tu-100 did not persist in the ectorhizosphere of oilseed rape. Populations of this isolate decreased from 8.5×10 colony-forming units (CFU) per seed to approximately 10 CFU in the plant ectorhizosphere within 30 days of sowing in autoclaved soil. Introduction Sclerotinia sclerotiorum (Lib.) de Bary is distributed worldwide and causes disease on oilseed rape and many other important crops (Boland and Hall 1994; Purdy 1979). Serious yield losses can result with annual losses to S. sclerotiorum on oilseed rape (Brassica napusL.), sunflower (Helianthus annuus L.), and soybean [Glycine max (L.) Merr.] estimated at more than U.S. $ 60×10 (Lu 2003). This pathogen overwinters as sclerotia in soil and generally infects plants as mycelia originating from eruptive germination of sclerotia in soil or as airborne ascospores that directly penetrate host tissue (Lu 2003). Diseases caused by this pathogen are typically initiated on above-ground plant parts (Abawi and Grogan 1979). On oilseed rape, stem lesions develop from the soil line or axils of branches or leaves. Lesions can eventually expand to girdle the stem and kill the plant (Nyvall 1979). Oilseed rape is the major oilseed crop in the People’s Republic of China, with approximately 70×10 ha in production (Zhao and Meng 2003). Crop rotation to nonsusceptible hosts and fungicide application are the prominent methods for control of diseases caused by S. sclerotiorum (Lu 2003; Yu and Zhou 1994). Although several fungicides are available, these chemicals can be expensive, ineffective, and hazardous (Lu 2003). There are also concerns regarding fungicide resistance in populations of S. sclerotiorum (Gossen et al. 2001). Traditional oilseed rape breeding programs for disease resistance have been hampered by a limited gene pool (Lu 2003). Furthermore, selection of oilseed rape cultivars with low seed glucosinolate content has become a priority for plant breeders. Glucosinolates are secondary metabolites present in crops of the Brassicaceae family, some of which produce toxic compounds when degraded (Zhao and Meng 2003). It is suspected that low glucosinolate levels in seeds lead to reduced resistance to S. sclerotiorum and other pathogens (Liu 2000). Certain degradation products of glucosinolates, especially hydrolysates of indoyl glucosinolate, have been reported to defend plants from insects and fungal pathogens on leaves (Brown and Morra 1997; Mithen X. Hu . M. Jiang . Y. Zhang Key Laboratory for Genetic Improvement of Oil Crops, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, 430062, People’s Republic of China D. P. Roberts (*) Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, United States Department of Agriculture–Agricultural Research Service, Beltsville, MD 20705, USA e-mail: [email protected] Tel.: +1-301-5045680 Fax: +1-301-5046491 1992). Because of these health and environmental concerns, and the current breeding trends, alternative control measures are needed for disease caused by S. sclerotiorum on oilseed rape in the People’s Republic of China. Biological control of diseases caused by S. sclerotiorum receives considerable attention. Coniothyrium minitans (Campbell) and several other biological control agents suppress various diseases caused by S. sclerotiorum on a number of crops (Adams and Fravel 1990; Escande et al. 2002; Huang et al. 1993; Li et al. 2003; McQuilken et al. 1997). Commercial products based on C. minitans, developed for the control of S. sclerotiorum on oilseed rape and other crops, are marketed in Europe (de Vrije et al. 2001). These products are not used in the People’s Republic of China, possibly due to high cost at current exchange rates. Consequently, there is a need for biological control agents for the suppression of S. sclerotiorum on oilseed rape that can be marketed for use in China. We report here decreased incidence of disease caused by S. sclerotiorum and improved plant vigor of oilseed rape in field trials in China with treatments containing Bacillus subtilis (Ehrenberg) isolate Tu-100. Materials and methods Bacterial and fungal isolates B. subtilis Tu-100 was isolated from an oilseed rape rhizosphere (Hu et al. 1992) and shown to inhibit a number of bacterial and fungal plant pathogens on agar media (Hu, unpublished data). Isolate Tu-100 was cultured in Luria– Bertani (LB) broth or agar with or without 30 μg ml kanamycin. B. subtilis Tu-100 is naturally resistant to 30 μg ml kanamycin. Strain Tu-100(pUC18) was constructed by introduction of plasmid pUC18 (Promega Corp.,Wuhan, People’s Republic of China), containing lacZ, into isolate Tu-100 by electroporation (Chassy et al. 1988). Strain Tu-100(pUC18) was similar to strain Tu-100 in growth rate, colony morphology, in vitro inhibition of S. sclerotiorum Ss-1, and inhibition of isolate Ss-1 in detached-leaf assays (data not shown). Strain Tu-100(pUC18) was cultured on LB broth or agar plus 50 μg ml ampicillin. Bacterial broth cultures were incubated at 150 rpm and 28°C. S. sclerotiorum Ss-1 was isolated from a sclerotium formed on oilseed rape. Isolate Ss-1 was maintained on potato dextrose agar (PDA) at 4°C. All microorganisms were obtained from the culture collection of the Plant Protection Laboratory, Oil Crops Research Institute (Chinese Academy ofAgricultural Sciences,Wuhan, People’s Republic of China). In vitro inhibition assays For in vitro inhibition assays, one disk (9 mm diam.) of isolate Ss-1 (from a PDA plate) was added to the center and four disks (9 mm diam.) of Tu-100 (from a PDA plate) were added to the periphery of each fresh PDA plate. These PDA plates were incubated at 22°C for 5 days prior to measuring the zone of inhibition of hyphal growth. For this, the distance between the bacterial colony and the edge of the Ss-1 mycelium was determined. The experiment was performed three times with five replicate PDA