| Rapeseed is an important oil crop and cash crop, and it is necessary to improve the yield of rapeseed under the situation of decreased rapeseed planting area. The occurrence of sclerotinia seriously hinders the production of repeseed, and the disease incidence could be up to 80% in some cases. It is difficult to control the disease due to soil-borne and airborne characteristic. As alternatives to chemical control methods, biological control methods have been received a lot of concern. Previous results from our lab suggested that application of bio-organic fertilizers could decrease some soil-borne disease incidence, and made some significant achievements. However, little was known about the effect of bio-organic fertilizer applied on rapeseed production. In this study, two bacterial strains, NJZJSB3 and NJZJSA2, which showed strong inhibition against S. sclerotiorum, were screened out, and the taxonomy of both strains were identified. The potential for biological control of rapeseed stem rot and mechanisms involved in biocontrol were studied. In particular, strain NJZJSA2 were chose for a second solid fermentation to produce specific bio-organic fertilizer(BOF). The effect of BOF applied on growth promotion and sclerotinia stem rot of rapeseed were studied. The results obtained are listed as follows:1. Several strains with inhibitory effects against S. sclerotiorum were isolated from the rhizosphere soil of healthy cruciferae plant from Tzu-chin mountain among which Strain NJZJSB3 and NJZJSA2 performed best in suppression of S. sclerotiorum in vitro. The diameter of culture filtrate inhibition zone on S. sclerotiorum were 27 mm and 44 mm,respectively. Both of the strains exhibited broad-spectrum antimicrobial activity against phytopathogens including F. oxysporum f.sp. niveum, F. oxysporum f.sp.cucumuerimu, F.oxysporum f.sp. melonis, Verticillium dahliae Kleb, Rhizoctonia solani, F. oxysporum f.sp.cubense, F. oxysporum Schlecht. On detached leaves bio-control tests, strain NJZJSB3 and NJZJSA2 could efficiently protect rapeseed leaves from S. sclerotiorum infection. Based on the characteristics of the colony morphology, physiological and biochemical tests, strain NJZJSB3 and strain NJZJSA2 were identified as Bacillus amyloliquefaciens and Streptomyces albulus, respectively.2. Culture medium and fermentation conditions for antifungal substances production by strain NJZJSB3 and NJZJSA2 were optimized by single factor analysis. We found that the Landy culture medium was suitable for strain NJZJSB3 and fermentation conditions was as follows: culture temperature 35?, initial pH 7.0, culure volume 50 mL in 250 mL flask, rotary speed of 170 r-min-1, fermentation time 60 h. The diameter of inhibition zone of strain NJZJSB3 fermentation filtrate was 36.67 mm. Strain NJZJSA2 was cultured in CGA culture medium and fermentation conditions was as follows: culture temperature 30?, initial pH 6.0, culure volume 100 ml in 250 ml flask, rotary speed of 170 r-min-1,fermentation time 7 d. The diameter of inhibition zone of strain NJZJSA3 fermentation filtrate was 46.83 mm. The antifungal substances produced by strain NJZJSB3 and NJZJSA2 were both stable in natural daylight, high temperature and resistant to protease. In greenhouse experiments, spraying cells suspension of strain NJZJSB3 and NJZJSA2 to rapeseed leaves could significantly inhibit development sclerotinia lesion at seedling stage,and the disease incidence were reduced 87.7% and 63.3%, respectively. Spraying culture filtrate of strain NJZJSB3 and NJZJSA2 were more efficiency in controlling disease lesion and the disease incidence were only 16.7% and 0%, respectively. The diameter of disease spot of infected leaves after treated by strain NJZJSB3 and NJZJSA2 were significantly smaller than the control treatment.3. The strain NJZJSB3 could produced siderophore,?-1,3-glucanase,protease. A total of 8 gene fragments of the expected size correlated with antibiotic biosynthesis, including surfactin, fengycin, iturin were efficiently amplified from strain NJZJSB3. The growth inhibition rate of the acid precipitation and methanol extracts from the culture filtrate against S. sclerotiorum was 71.1% in vitro assays. HPLC-ESI-MS analysis suggested that strain NJZJSB3 produced one group of ion peaks with mass difference of 14 Da at 1,030.6,1,044.7, 1,058.8, 1,072.8, 1,086.8 Da. HPLC analysis suggested that iturin A standard solution and the crude extracts had the same peaks at the same retention times. Concerning the literatures and our results, the compounds was identified as iturin analogs with different side chain length (-CH2-). Antifungal volatile organic compounds were identified by GC-MS. The detected volatile benzothiazole showed antifungal effects against S.sclerotiorum, respectively.4. Strain NJZJSA2 growing medium conditioned with deactivated S. sclerotiorum could enhance cell wall-degrading enzymes (chitinase, ?-1,3-glucanase, and protease)production. The enzyme activates in this medium were significantly (P?0.05) higher than that on glucose medium. The protein extracted by ammonium sulfate precipitation method could inhibit S. sclerotiorum mycelia growth. SEM analysis showed that the mycelia and sclerotinia cell wall collapsed and rupture and intracellular material leaked out. In the detached rapeseed leaves biocontrol experiment, the crude protein solution could inhibit S.sclerotiorum infection. The VOCs generated by strain NJZJSA2 significantly inhibited mycelial growth and sclerotinia germination of S. sclerotiorum. Twelve VOCs were identified based on GC/MS analyses. Among them, five compounds were purchased and used for the antifungal activity assay. The antifungal activity order of the five compounds was shown to be 4-methoxystyrene > anisole > 2-pentylfuran > styrene > tetradecane at the concentration level of 80 ?l/plate. SEM analysis showed that the pathogen hyphae were shriveled and damaged after treatment with 4-methoxystyrene. Optical micrograph results indicated the loss of cellular impermeability to trypan blue. We can infer that the antifungal activity of VOCs was achieved by destroying the cellular impermeability of fungal cell membrane.5. In order to study the growth promotion ability of strain NJZJSA2, the cells suspension of strain NJZJSA2 was irrigated to the root of rapeseed. The shoot and root biomass of bio-organic fertiliezer-treated plants were promoted by 45.0% and 30.4% when compared with the control treatment, respectively. The length, surface area, volume of roots was also significantly increased. The growth promotion factor of strain NJZJSA2 were analyzed by qualitative test. Results showed that the strain NJZJSA2 have the phosphate-solubilizing ability and could secrete siderophore. High-performance liquid chromatography (HPLC) analysis in vitro suggested that strain NJZJSA2 could produce plant growth hormone, indole acetic acid (IAA). Strain NJZJSA2 may colonize the rapeseed rhizosphere by using rapeseed root exudates. Four organic acids (oxalic acid,malic acid, citric acid and succinic acid) were identified from rapeseed root exudates. All the organic acids exhibited different and significant stimulation effects on mycelial growth and conidial germination of strain NJZJSA2 in vitro. In greenhouse experiment, nursery application of BOF contain strain NJZJSA2 significantly increased the biomass of the rapeseed plants compared with the treatments applied with organic fertilizer (OF) or with chemical fertilizer (CF). Relative to the control, BOF treatment increased fresh shoot and root biomass of rapeseed plant by 31.8% and 25.5%, respectively, while there was no significant difference between OF and CF treatment biomass. The activities of urease,catalase, sucrase and acid phosphatase of rhizosphere soil of BOF treatment were increased by 18.4%, 13.5%, 70.6% and 45.4%, respectively. They were increased by 6.69%, 6.97%,69.8% and 33.2%, respectively in the OF treatment. The results of the second greenhouse experiment showed that the fresh shoot biomass of cells suspension of strain NJZJSA2 (A2)treatment were increased by 11.7% when compared with the blank control (CK), the fresh shoot biomass of BOF were increased by 25.7% when compared with OF treatment. These results suggest that strain NJZJSA2 has the ability to promote rapeseed growth, and the BOF may enhance the ability of strain NJZJSA2 in soil.6. In field experiments, the fresh shoot biomass of CF, OF and BOF treatments were significantly higher than CK treatment. The fresh shoot weight, plant height, and stem width in the BOF treatment were increased by 67.8%, 32.6% and 12.67%, respectively,when compared with CF treatment at the first sampling time; these fresh shoot biomass data were increased by 38.4%, 17.0% and 19.5% at the second sampling time; and by 22.3%,11.3% and 18.5% at the third sampling time, respectively. The fresh shoot biomass in the OF treatment and CF treatment showed no difference during the three sampling times. BOF treatment altered the microbial community structure and improved plant health. The counts of bacteria and actinobacteria in the rhizosphere soils were significantly increased in the BOF treatments, whereas the fungi in the soil were decreased when compared with CF treatments. However,the count of bacteria, actinobacteria and fungi in the OF treatments were all increased. The rapeseed yield was negatively correlated with sclerotinia disease incidence. The yield of BOF, OF, CF and CK treatments were 241.9?221.1?215.2 and 96.9 kg/mu, while the disease incidence of each treatment were 42.7%,53.0%,46.3% and 58.0%. In the second field experiment, the sclerotium disease incidence of the BOF+spraying strain NJZSA2 cells suspension (BOF+A2) and BOF was 40.0% and 55.33%, respectively. The CF+ spraying strain NJZSA2 cells suspension (CF+A2) and CF was 52% and 68%, respectively. The rapeseed yield of BOF+A2, BOF, CF+A2 and CF treatment were 284.8?181.9?240.8 and 155.8 kg/mu,respectively.In conclusion, strain NJZJSB3 and NJZJSA2 have the ability to suppressing S.sclerotiorum infect rapeseend leaves. We found these strains could excretion several antifunal substances. Our studies showed that the BOF containing strain NJZJSA2 could decrease the sclerotinia stem rot disease incidence. Combination of using BOF and spraying strain NJZJSA2 could effectively inhibit sclerotinia stem rot, and promote the growth of rapeseed plants. Results in this study would be useful in the developing new methods to better control sclerotinia stem rot of rapeseed. |
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