| Phytophthora parasitica var. nicotianae, a soilborne pathogen, causes tobacco black shank. A lot of methods have been used to prevent the disease, among which biocontrol has been proved to be most promising. Biological control is one of the important measures for overcoming soil-borne diseases, because of its effectiveness and environment-friendly use. Screening antagonists against pathogens is the vital work for the biocontrol of plant diseases.In this study, an antagonistic strain of Paenibacillus polymyxa C5effective against Ph. parasitica var. nicotianae, was isolated from the rhizosphere soil of a healthy tobacco plant grown in the field strongly attacked by tobacco black shank. A novel bio-organic fertilizer (BOF) was developed by inocalating P. polymyxa C5and doing the second solid fermentation. Pot experiments were carried out to evaluate the biocontrol effect of this BIO against tobacco black shank, and the related mechanisms were also investigated. The main results obtained are listed as follows:1. Pot experiments revealed that the application of bio-organic fertilizer containing strain C5(BOF) could decrease the incidence of tobacco black shank by80%compared with control (CK2) in which neither C5strain nor organic fertilizer were used in continuous cropping soil (diseased soil). The application of BOF could influence the microbial community of rhizosphere soil. The population of bacteria and actinomyces was increased by2.4times and4.4times compared with control, respectively.The fresh weight, dried weight and height of tobacco in BOF were increased by about1.8times,1.7times and1.4times, respectively, compared with CK2. The fresh weight and plant height of tobacco in BOF were increased by about1.4times and1.3times compared with those of CK1(healthy soil). The fresh weight, dried weight and plant height of tobacco in BIO were increased by about1.4times,1.4times and1.2times compared to those of OF. These results indicate that the bio-organic fertilizer could not only control the tobacco black shank, but also promote tobacco plant growth.2. The extracts of P. polymyxa C5had a significantly antifungal activity against the pathogen. Compounds with antifungal activity were detected in the extracts, which were stable at neutral pH and low temperatures, and resistant to proteinase. One peak of the crude compounds with antifungal activities was detected by high-performance liquid chromatography (HPLC). Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis showed that the mass spectra of the peaks were characterized by two series of homologous ion peaks, one with molecular weights of883.5and897.5Da and the other with molecular weights of947.5and961.5Da. The two series of compounds were identified as Fusaricidins.3. Six kinds of phenolic acids, including chlorogenic acid, caffeic acid, syringic acid, ferulic acid, salicylic acid and cinnamic acid were detected in the root exudates of tobacco by HPLC. However, chlorogenic acid, caffeic acid and syringic acid were excreted when the tobacco roots were excited by C5or the pathogen. The growth of Phytophthora parasitica var. nicotianae was promoted by low concentration of chlorogenic acid, caffeic acid and syringic acid. The main organic acids of root exudates from tobacco plants were oxalic acid and malic acid. The growth of stain C5was promoted in the presence of40μM oxalic acid and malic acid of40μM. Furthermore, C5showed the highest chemotaxis reaction response to these organic acids.4. Colonization assay indicated that the green fluorescent P. polymyxa C5cells mainly colonized the root tips and elongation zone of the root surfaces, as well as root hairs. High densities of the introduced GFP-tagged C5strain could be recovered on the root surface and in the rhizosphere soil reaching approximately106cfu g-1root and106cfu g-1soil six days after the inoculation.In conclusion, our studies showed that the bio-organic fertilizer, appropriate combinations of organic fertilizer with the antagonistic strain P. polymyxa C5used in the present study, could effectively suppress the tobacco black shank. To reduce the losses of tobacco production caused by Phytophthora parasitica var. nicotianae, the mechanism of the biocontrol of tobacco black shank should be further studied. |
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