Biological Control Of Endophytic Bacterium Strain Em7Against Sclerotinia Sclerotiorum On Oilseed Rape

Oilseed rape(Brassica campestris L.) is the major oil crop in China, with a production area of approximately70×106ha. Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is one of the important diseases on oilseed rape in the world. In China, the disease incidences were10-20%in general, but reach over80%in serious land or serious year. Conventional methods for controlling Sclerotinia disease such as chemical and cultivating control measures have been applied in different countries for many years. However, conventional breeding programs to increase resistance in rape to Sclerotinia disease have been less effective because of the restricted gene pool; and because of the adverse effects on non-target organisms and environment, the use of fungicides have been limited. Therefore, the relatively unreliable control with traditional methods and concerns about fungicides residues has prompted interest in biological control as an alternative strategy for disease management. Endophytes, living asymptomatically within plant tissues, have beneficial effects on host plants. In recent years, endophytic bacteria have gained increasing scientific interest, but few studies were reported about biocontrol of oilseed rape Sclerotinia stem rot by endophytic bacteria. In preliminary studies, we detected that the endophytic bacterial strain Em7strongly inhibited S. sclerotiorum mycelium growth and sclerotia formation in vitro. Then, the biocontrol effect and mechanism were studied in this paper. The main results were as follows:1. Biocontrol efficacies of strain Em7to S. sclerotiorum were detected in greenhouse and in field trials. In the greenhouse, the culture filtrates and cell suspensions of strain Em7displayed effective biocontrol activities in the seedling and blossom stage. The biocontrol activities were influenced by the time and concentration of spraying strain Em7. The control efficacy was higher when strain Em7was applied prior to inoculation of the pathogen than after inoculation. Three-year field trials (2009-2011) showed that twice sprays of strain Em7cell suspension at blossom stage significantly reduced disease incidence and severity. The control efficacies were53.8%,71.8%and70.5%, respectively in2009,2010, and2011. There was no significant difference in control efficacy among treatments with strain Em7and the fungicides Carbendazim or Tebuconazole.2. Strain Em7was identified as Bacillus subtilis (Ehrenberg) Cohn on the basis of morphology, physiological and biochemical characteristics,16S rDNA sequence analysis and Biolog microbial automatic identification system.3. The strain Em7colonization in rape plant was detected by rifampicin-labeled Em7. The results showed that bacteria could colonize into stems, leaves and roots, but the colonization of mutant strain Em7could be determined by different treatment. Bacteria could largely colonize into stems and leaves after spraying1d, the number of mutant strain Em7were4.0×105CFU·g-1and9.6×105CFU·g-1, respectively, in leaves and stems. Then the quantity gradually decreased. The labelld-strain could be recovered from stems and leaves of plants even after28days, the quantity is up to3.1×102CFU·g-1in stems. The mutant strian could be detected in roots and stems after root drench treatment. The biomass of mutant strain Em7in roots and stems could be reached5.6×106CFU·g-1and4.6×105CFU·g-1after3d. Although the quantity gradually decreased, the labelled strain could be still detected in stems and roots after28d.4. The effects of applying biocontrol agent strain Em7on the oilseed rpae soil microbial communities was evaluated by Biolog metabolic fingerprinting method. The profiles derived from Biolog ECO plates indicated higer substrate richness, average well color development (AWCD, microbial activity) were influenced by strain Em7. The values of AWCD and Shannon functional diversity index were higher treated with strain Em7than untreated.5. It was concluded that the induced resistance is one of the biocontrol mechansim of endophytic bacteria Em7against S. sclerotiorum. The culture filtrate and cell suspensions of strain Em7was applied prior to inoculation of the pathogen in the seedling and blossom stage, the resistance of oilseed rape was induced to S. sclerotiorum. After the treatment of strain Em7, the activity of POD, PPO and PAL had obviously rised in certain period of time, which had significant difference to the contrast.6. Light and scanning electronic microscopy studies revealed that in the presence of strain Em7, morphology of mycelial cell of S. sclerotiorum showed leakage, deformation, and disintegration of hyphal cytoplasm. The ultrastructure of mycelial cell was investigated under transimission electronic microscopy. The results showed that strain Em7caused increasing of vacuole in the cytoplasm, disordered of the orgarelles, rupture of the cell wall, and leakage of the cytoplasma. The effects of strain Em7on the infection and extension of S. sclerotiorum in the rape leaves were observed under scanning and transmission electronic microscopy. Treated with strain Em71d before inoculation, the mycelia couldn't form the infection structure on the sueface of leaves, and the mycelia were irregularlly swollen and collapsed. Treated with strain Em71d after inoculation, the infected cushion was destroyed, and the hyphae were collapsed and coated by film, the cytoplasm exosmosis. In Petri dish, strain Em7could inhibit not only mycelial growth but also sclerotia germination. However sclerotia germination was obviously influenced by concentration of strain Em7cell suspension.7. The optimum fermentation culture medium of strain Em7was optimized at shake flask. The results of single-factor experiment and the orthogonal test showed that the optimum medium was bran2.0%, beef extract1.0%, and MnSO40.15%. The maximum antibiotic activity was achieved at the initial pH7.0, medium volume of50ml in250ml flasks, rotary speed of150rpm, temperature28℃and inoculation volume of1%. The antifungal substance was studied by ammonium sulfate precipitation and Gel filtration chromatography. The main active substance of strain Em7as protein antibacterial substance had been definited. Crude protein extracted by30%saturation ammonium sulfate was stable to thermal, acid and alkali. Furthermore, the antifungal protein also showed a broad antifungal spectrum on mycelium growth and conidia germination of numerous important plant pathogenic fungi. Light microscopic observations revealed that the antifungal protein caused morphological alterations including increased branching, swelling, and shriveling of hyphae as well as collapse of cytoplasm.