| In this paper, pathogen in bee pollen were identified by general PCR, and the dynamics changes of pathogen inhibited by water-soluble chitosan solution were detected by fluorescent real-time quantitative PCR (RT-qPCR). The inhibitory concentration of chitosan solution on pathogen, especially on Ascosphaera apis in rape bee pollen was studied. Results are as follows:I. Nine fungus strains (F1-F9) and twelve bacterium strains (B1-B12) were isolated from rape bee pollen. By morphological identification, strains F1 and F5 were primarily putative to terrene mold Aspergillus terreus, strain F4 to black mold Aspergillus niger, strains F2, F3, F6 and F7 to rhizome mold Rhizopus, strain F8 to honeybee cocci Ascosphaera apis, strain F9 to yellow mold Aspergillus flavus and most of strains B1-B12 to bacilli Bacillus subtilis.II. Based on the results of morphological identification, the gDNAs of six strains including Bacillus subtilis, Aspergillus flavus, Aspergillus, Rhizopus, Aspergillus niger and Ascosphaera apis were chosen to design primers, respectively, for further identification by general PCR. The sequence of PCR products were assigned to the database in NCBI for similarity, they were archived in the categories of F1 and F5 as Aspergillus terreus, F4 as Aspergillus niger, F2, F3, F6 and F7 as Rhizopus, F8 as Ascosphaera apis, F9 as Aspergillus flavus, B1-B12 except for B6 and B8 as Bacillus. III. The inhibition of water-soluble chitosan at different concentrations on pathogen that had been isolated from bee pollen were detected by general PCR and by real-time fluorescence quantitative PCR, respectively. The results showed that the dynamics changes of total DNA before or after inhibition could not be presented by the DNA brightness of general PCR products that resulted from DNAs of mixed strains as a template. The CT value given by the RT-qPCR indicated that there existed such an effect of water-soluble chitosan on 6 kinds of pathogen, the higher the concentration was, the stronger inhibition would be.IV. The results from the inhibitory test of water-soluble chitosan solution at different concentrations gradients on fungi (F1-F9) and bacteria (B1-B12) in bee pollen showed that there was a better inhibitory effect on fungi. Various strains of fungi had different resistance to chitosan, they were F7 and F8 (>0.02g/mL), F1 and F3 (>0.04g/mL), F4 (>0.06g/mL), F2, F6 and F9 (middle-resistant), F5 (strong-resistant) by a sequence of concentration at which their inhibitory rate reached to more than 50%, respectively. Various strains of bacteria had different sensitivity to chitosan, they were B4 (0.003125g/mL), B2, B3, B5, B6, B10, B11 and B12 (0.00625g/mL), B8 (0.0125g/mL), B1, B7 and B9 (0.025g/mL) by a sequence of their minimum inhibitory concentration (MIC), respectively.V. Honeybee cocci Ascosphaera apis (the main pathogen of bees chalkbrood disease) which had a weakest resistance to chitosan was chosen for further antiblastic test. The results showed that mycelium could not grow on the plate at a concentration of more than 0.04g/mL but could slowly grow, compared with that in control group, at a concentration of less than 0.04g/mL after culture for 48h. No colony was found to grow on the plate at a concentration of more than 0.08g/mL after 72h. The inhibitory growth rate varied with the concentration of chitosan after culture for 3d, 5d and 7d, it would be 60% when the concentration was greater than 0.04g/mL, but the percent subsequently went slow; the inhibitory effect decreased with the time length at the same concentration. |
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