| Plant beneficial microorganisms can improve plant growth status and promote plant growth through their own life activities.Biological fertilizers based on them can replace chemical fertilizers and pesticides in a green way.Applying biological fertilizers in agricultural production can reduce the use of chemical fertilizers and pesticides,thereby reduce its pollution to the ecological environment.Plant tissue and rhizosphere are the gathering places of plant beneficial microorganisms.This thesis isolated and screened plant beneficial microorganisms from tea tree leaf tissue and rhizosphere soil,and explored their biological characteristics through the following work.(1)The bibliometrics method was used to systematically evaluate the relevant literatures in the field of bioferfertilizer.An in-depth analysis of the characteristics of12880 journal articles published from 1980 to 2022 found that the number of journal articles related to biofertilizers increased exponentially.Developing countries(India,Brazil,Pakistan,Iran,etc.)do not produce enough chemical fertilizers to meet agricultural needs,so developing countries put more effort into biofertilizer research.This is probably the main reason why developed countries account for only 20% and 30% of the top five and ten countries in terms of journal articles published.The rapid growth in annual journal articles published in India and China following the enactment of biofertier-related policies suggests that government policy is one of the main drivers of biofertier-related research.The authors of the three stages of the network co-discovery findings: the first stage of the research focus on the use of biological fertilizer for nitrogen fixation,so as to promote crop growth and improve crop yield;In the second stage,researchers began to pay attention to the effect of biofertilizers on environmental stress.In the third phase,the application of biofertilizers in addition to promoting plant growth was extensively investigated.It was found that the two most prominent key words were "Rhizobacteria" and "Endophyte",indicating that rhizosphere and plant tissue were the main locations for bioferters to survive and function.The content analysis showed that the main action mechanisms of bioferfertilizer were: improving and supplying plant nutrients,regulating plant growth state,and improving soil conditions and microbial community.(2)Bacillus velezensis can secrete lipopeptide antibiotics with excellent stability,and it is a biocontrol strain with great potential for development.A biocontrol bacterium Z-1was isolated and screened from the leaf tissue of healthy tea tree.In the plate confrontation test,it was found that strain Z-1 could cause the hyphal tip of plant pathogens to expand and rupture,thus inhibiting the hyphal growth.Strain Z-1 was identified as Bacillus velezensis by morphology and molecular biology.The growth curve showed that strain Z-1 had a short retardation period and was prone to rapid propagation in a short time.The bacteriostatic ability of the aseptic fermentation broth of strain Z-1was enhanced gradually with the extension of culture time,and the bacteriostatic activity of the aseptic fermentation broth was not affected by temperature and pH.In the oil discharge test and xylene emulsification test of the sterile superneant,it was found that it could not only form an oil discharge ring on the surface of the oil layer,but also emulsify xylene,indicating that the bacteriostatic substances existed both hydrophilic and oilphilic groups.Combined with previous studies,it was speculated that the bacteriostatic substances might be lipopeptide substances.The crude antimicrobial extracts were successfully extracted from the supernatant of fermentation by acid precipitationmethanol extraction method,and it was found that the substance still had antibacterial effect at 0.01 mg/mL.Thin layer chromatography analysis showed that there were two kinds of substances containing amino acid residues in the crude extract.Fourier transform infrared spectrum analysis found that there were peptide bonds and fatty acid chains in the crude extract.Combined with the previous test,it was proved that the substance was a lipopeptide substance.Mass spectroscopic analysis showed that Kurstakin was the corresponding lipopeptide with an obvious peak(922 m/z)at 850-950 m/z.The pot experiment showed that Z-1 could improve the damage of Fusarium Fujikura on wheat,and the effect was similar to that of carbendazim.(3)Soluble phosphorus is easy to form insoluble calcium phosphate with calcium ions in alkaline soil,which reduces the content of soluble phosphorus in soil and affects the normal growth of plants.A fungal strain JL-1 was isolated and screened from rhizosphere soil of tea tree,which was highly capable of dissolving tricalcium phosphate.The strain was identified as Penicillium rubens by morphology and molecular biology.By detecting the dynamic changes of phosphorus content,pH value and organic acid content,it was found that the correlation coefficients between pH value and gluconic acid content,pH value and phosphorus content,and gluconic acid content and phosphorus content in fermentation broth were-0.991,-0.981 and 0.993,showing significant negative correlation,significant negative correlation and significant positive correlation,respectively.Electron microscope was used to observe the surface morphology of tricalcium phosphate,and it was found that compared with the control group(JL-1 strain was not inoculated),the large particles of tricalcium phosphate in the experimental group(JL-1 strain was inoculated)all disappeared,and only a few small particles of tricalcium phosphate were eroded by acid.It was preliminarily shown that the mechanism of phosphorus solubilization of JL-1 strain was to promote the dissolution of calcium phosphate by secreted gluconic acid.Pot experiments showed that JL-1 strain could also promote the release of phosphorus in tricalcium phosphate in soil,promote the growth of wheat,and significantly increase the root length(157.9%),plant height(136.4%),total fresh weight(142.9%)and top fresh weight(140.8%)of wheat,which has great potential to be developed into microbial phosphorus fertilizer.(4)Through the single factor test,it was found that the best carbon source for JL-1strain to dissolve phosphorus was glucose,the best nitrogen source was ammonium sulfate,and inorganic nitrogen sources(ammonium sulfate,ammonium chloride,ammonium nitrate and sodium nitrate)Phosphorus amount did not have a significant effect,which is also consistent with the conclusion that JL-1 strain dissolves phosphorus by secreting gluconic acid.Through the Plackett-Burman design,it was determined that the significant influencing factors were glucose content,tricalcium phosphate content,and temperature,and the optimal values of glucose,tricalcium phosphate,and temperature were determined to be 29.8 g/L,7.1 g/L,and 31.9°C.The verification test found that the amount of dissolved phosphorus was 1194.15 mg/L,which was consistent with the predicted value of the model,and it was nearly 3 times higher than that under the initial conditions. |
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