| Phosphorus is one of the main nutrient elements of plant growth and development.And the phosphorus in the soil is abundant in our country.However,a large amount of phosphorus in the soil can not be absorbed by the crops or effectively stored in the soil in the form of the invalid phosphorus,due to the specific physical and chemical properties of the soil and the excessive and inappropriate application of phosphate fertilizer,which resulted in Phosphorus waste and phosphorus pollution.Phosphorus-solubilizing bacteria(PSB)can convert ineffective phosphorus in soil into available phosphorus that can be absorbed and utilized by crops by secreting extracellular enzymes and releasing substances(organic acids,protons,hydroxyl ions,etc.).Therefore,separating high-efficiency solubilizing bacteria from the natural environment to reduce the use of chemical fertilizers and relieve the pressure on the environment is of great significance for green,environmentally friendly and sustainable modern agriculture.In this study,activated sludge from a phosphating plant in Hubei was used as a material.A phosphate-solubilizing bacterium that can efficiently dissolve solid calcium phosphate was selected and named MEL01.Biochemical and 16S rDNA identification showed that the strain MEL01 was incubated at 28 ? for 48 h on LB plates.The colonies were round and well-marginated.The pale yellow color was opaque and the surface was wet and viscous.The Gram stain was negative and the strains were negative.It can grow in 1%NaCl,pH=5 conditions,hydrolyze gelatin,D-fructose and D-galactose,and tolerate rifamycin SV,vancomycin,ammonia Qunan and other antibiotics,but it can not hydrolyze dextrin,inosine,glycerol.By constructing a phylogenetic tree of this strain,the strain MEL01 was identified as Burkholderia gladioli.The MEL01-activated broth was inoculated in a liquid calcium phosphate and liquid phosphate rock medium at inoculation of 2%,and the phosphorus-dissolving effect was measured.The results showed that the MEL01 dissolved calcium phosphate at 28 h.The soluble phosphorus content reached its maximum at 345.58 mg/L.At 28 ? the soluble phosporus content of MEL01 dissolved phosphate rock reached its maximum at 107.69 mg/L at 96 h.The mechanism of phosphorus dissolution of MEL01 was detected by HPLC.The results showed that MEL01 could secrete organic acids such as oxalic acid and gluconic acid during the process of dissolving insoluble inorganic phosphorus.In addition,after inoculating MEL01 in soy lecithin liquid medium,the degradation of soybean lecithin reached the maximum at 36 h and the soluble phosphorus content was 41.43 mg/L at 36 h.MEL01 was inoculated into chitosan fermentation medium with chitosan concentration of 1%,and its growth curve and enzyme activity curve were determined.The results showed that MEL01 reached a stable phase at 20 h and the stability period was longer until 72 h.At 24 h,chitosanase activity reached a maximum of 4.03 U/mL.Similarly,the growth curve and enzyme activity curve of chitin with 1%hydrolysis of MEL01 were also determined.At 60 h and 28 ?,the chitinase activity reached a maximum of 17.36 U/mL.Finally,the fungus antagonistic activity of MEL01 strain was tested.The results showed that:MEL01 could significantly inhibit the six species of Physalospora spp.,Phytophthora indicus,Rhizoctonia solani,Pythium multiformis,Botrytis cinerea,and Fusarium oxysporum The growth of phytopathogenic fungal hyphae indicated that MEL01 had great potential in biological control applications.The total DNA of MEL01 was extracted and subjected to genome frame sequencing.The chitosanase protein sequence was compared with the chitosanase sequence on NCBI,and the similarity to the protein sequence of the glycanase of B.gladioli CHB101,up to 99%;and the similarity of the chitosanase gene sequence is also high,initially identified as the same enzyme.On the filter paper,the MEL01 fermentation broth was used to germinate the seed,the results showed that the germination rate of the Chinese cabbage,Chinese cabbage,red radish and rape seed in the experimental group was 100.0%at 7 d,7 d,5 d and 7 d respectively.The corresponding germination rates of the blank control and the positive control were the same.They were 40.0%,60.0%,50.0%,and 60.0%,respectively,indicating that MEL01 fermentation broth can effectively promote the germination of Chinese cabbage,Chinese cabbage,red radish,and rape seed.The experimental results of MEL01 chitosan fermentation solution on the growth of Chinese cabbage showed that the plant height increased by 74.73%,the stem diameter increased by 183.33%,the root length increased by 4.51%,and the fresh weight increased by 2376.00%after 45 days of planting,so it had a significant role in promoting growth.By measuring the available phosphorus content in soil during the growth-promoting experiments,the experiment showed that the available phosphorus content in the experimental group reached the highest at 35 days,which was 35.44 ±0.95 mg/kg,which was significantly higher than that of the corresponding control group at 15 days.The highest value was 27.65 ± 0.33 mg/kg,which showed that the MEL01 strain could effectively dissolve the phosphate rock,increase the soil available phosphorus content,and thus promote the growth of pakchoi.After the stimulation experiments,denaturing gel gradient electrophoresis(DGGE)experiments were performed on the soil of the experimental group,the negative control group,and MEL01 bacteria.Experiments showed that the soil of the experimental group appeared corresponding to the same position of the MEL01 bacteria.There was no corresponding band in the soil of the negative control group,and there was a consistent band in the soil of the experimental group and the negative control group at the same place,indicating that the MEL01 chitosan fermentation liquid was applied to the soil of the experimental group.After that,there was no drastic change in the entire soil microbial system.The soil samples from the original soil and negative control group,positive control group(system 3)and experimental group were analyzed for microbial diversity on the 15th day.The results showed that the soil of the experimental group could be maintained after applying the MEL01 chitosan fermentation solution.The stability of the soil microbial environment can also enhance the fertilizer efficiency by enhancing the activity of the phosphate-solubilizing bacteria group,thereby promoting the growth of the Chinese cabbage,indicating that the MEL01 chitosan fermentation liquid can be applied as an ideal and efficient biofertilizer.The effect of MEL01 chitosan fermentation broth on the growth of field-grown pakchoi was tested.The experiment showed that the growth rate of pakchoi's fresh weight reached 100.15%in the experimental group treated with MEL01 chitosan fermentation broth.Thus,the MEL01 strain is a highly effective phosphorus-solubilizing bacterium with a multifunctional function.It can efficiently dissolve insoluble inorganic phosphorus,degrade soybean lecithin,and secrete extracellular enzymes to hydrolyze chitosan and chitin.It can inhibit the growth of phytopathogenic fungi and promote the germination of crop seeds;it can be used as a biofertilizer to promote the growth of Chinese cabbage.Therefore,the multifunctional strain MEL01 is expected to be applied in the realization of green,sustainable development of ecological agriculture. |
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