| In this study,13 kinds of medicinal plants in Gansu province were used as materials to isolate endophytic and rhizosphere bacteria by conventional dilution coating plate method.The growth promoting ability of the strain was preliminarily detected by nitrogen fixation,phosphorus solubilization,IAA production and bacteriostatic function.The stability of bacteriostatic substances of the five selected functional strains was determined under p H,temperature,UV irradiation and storage conditions,which laid a foundation for the study of bacteriostatic mechanism.Five functional strains were identified by conventional techniques and 16S r DNA sequence molecular techniques to determine their phylogenetic status.The bacteriostatic substances of 5 functional strains were extracted preliminarily,and the bacteriostatic mechanism of the strains against pathogenic fungi was observed and analyzed by microscopic technique.In this study,several functional strains with nitrogen fixation,phosphorus solubilization,IAA production and bacteriostatic ability were isolated,which laid a foundation for the research and development of bacterial fertilizer and bacteriostatic agent for specific medicinal plants.The main results are as follows:1.The conventional dilution coating plate method was used to isolate strains from the rhizosphere and body of 13 medicinal plants.The results showed that:in this study,1169 strains were isolated from the rhizosphere soil of 13 medicinal plants,and the average bacterial density was 1.05×108 cfu·g-1.The number of endophytic bacteria in different stages of 13 medicinal plants was in the order of plant growth flourishing stage>plant growth and harvesting stage>plant growth and seedling stage,and the number of endophytic bacteria in different parts of the plant was in the order of root>stem>leaf.The average bacterial population density of 13 medicinal plants was 3.84×105 cfu·g-1.2.The rhizosphere growth-promoting strains were screened by nitrogen fixation,phosphorus solubilization,IAA production and bacteriostatic function.The results showed that:347 nitrogen-fixing strains,78 phosphorus-soluble strains,699 IAA producing strains and 18 antagonistic strains were isolated from the rhizosphere soil of 13 medicinal plants,accounting for 46.79%,6.67%,59.79%and 1.37%of the total rhizosphere respectively.A total of 72 rhizosphere strains with stable nitrogen fixation ability were screened.There were 12 rhizosphere strains with stable phosphorus solubility,the dissolved inorganic phosphorus content ranged from 1.21mg·L-1 to 38.87 mg·L-1.84 rhizosphere strains produced more IAA,and the secretion ranged from 6.28 to 46.42 mg·L-1.The antibacterial activity of 18 rhizosphere strains ranged from 21.51%to 68.21%.Five rhizosphere strains,DST21,DST27,WTT25,HQT43 and DT32,had different degrees of antibacterial activity against seven pathogenic fungi,such as Fusarium acuminatum and Botrytis cinerea.3.Endophytic growth-promoting bacteria were preliminarily screened by nitrogen fixation,phosphorus solubilization,IAA production and bacteriostatic function.The results showed that:318 strains of nitrogen-fixing bacteria,60 strains of phosphorus-soluble bacteria,589 strains of IAA producing bacteria and 14 strains of antagonistic bacteria were isolated from 13 kinds of medicinal plants,accounting for31.58%,5.69%,58.29%and 1.39%of the total number of isolated endophytic bacteria respectively.48 endophytic bacteria with stable nitrogen fixation ability.17endophytic bacteria with stable phosphorous dissolving ability,the amount of inorganic phosphorus solubilizing ability of endophytic bacteria ranged from 2.54mg·L-1to 49.37 mg·L-1.The 62 endophytic bacteria produced more IAA,with the secretion ranged from 5.10 to 56.38 mg·L-1,6 endophytic bacteria with the secretion of more than 40 mg·L-1,9 endophytic bacteria with the secretion ranged from 20 to 40mg·L-1,and 47 endophytic bacteria with the secretion of less than 20 mg·L-1.The antimicrobial activity of 14 endophytic strains ranged from 18.74%to 53.01%,and the three endophytic strains DSG3,DSY6 and KDG5 all had different degrees of antimicrobial activity against F.oxysporum,F.solani and F.equiseti.4.By treating the supernatants of the 5 functional strains differently,the stability of their antibacterial substances was studied.It was found that the antibacterial substances of the 5 functional strains were stable relatively to ultraviolet light and storage time,and would not be affected by long-term ultraviolet irradiation.The antibacterial substances of the 5 functional strains were stable relatively to temperature and p H,and the supernatants of the 5 functional strains were treated at28°C to 60°C,which basically had no effect on the stability of the antibacterial substances.After p H 6～8 treatments,the stability of the antibacterial substances remained basically unchanged.5.By studying the effects of fermentation supernatant of 5 functional strains with different soaking time and concentration on the germination of Astragalus membranaceus seeds,the germination rate,germination index,germination potential and average germination speed of 5 strains on Astragalus membranaceus seeds were determined as follows:The optimal soaking time of the supernatants of the five functional strains DST21,DST27,WTT25,HQT43 and DT32 were 4 h,6 h,4 h,8 h and 4 h,respectively.The optimal soaking concentration of DST21,DST27 and WTT25 supernatants was 9.45×107 cfu·m L-1,9.05×106 cfu·m L-1and 8.95×106cfu·m L-1,and the optimal soaking concentration of HQT43 and DT32 was 120 h.6.Combined with morphological characteristics,physiological and biochemical characteristics and 16S r RNA molecular identification,the five functional strains of strains DST21,DST27,WTT25,HQT43 and DT32 were identified respectively as Brevibacillus brevis,Bacillus pumilus,Paenibacillus polymyxa,Streptomyces albidoflavus,and Streptomyces kanamyceticus.7.According to the principle of similar compatibility,organic solvents of different polarities were used to extract antibacterial substances,and the enriched polar segments of antibacterial substances and active ingredients of 5 functional strains were determined:Chloroform should be used as solvent for crude extraction of DST21 and DST27 bacteriostatic substances,and acetone should be used as solvent for crude extraction of WTT25,HQT43 and DT32 bacteriostatic substances.The bacteriostatic substances of HQT43 and DT32 were polar substances,and the polarity of DT32 bacteriostatic substances was weaker than that of HQT43.Using microscopic techniques,the mechanism of action of the strains on pathogenic fungi was observed and analyzed:The three strains of DST21,DST27 and WTT25 had basically the same antibacterial effect on pathogenic fungi,with abnormal growth,malformation and thinning of mycelia,internode shortening and thickening,uneven thickness,contraction of cell protoplasm,mycelia fracture and disintegration.HQT43 and DT32strains had basically the same antibacterial effect on pathogenic fungi.The middle and top of mycelia expanded,twisted or malformed,forming separation,the protoplasm in mycelia gathered into blocks,resulting in mycelia cell enlargement and deformation,body wall dissolution,and finally mycelia lysis. |
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