Colonization And Growth-promoting Mechanism Of Phosphate Solubilizing Bacteria YL6 In Chinese Cabbage

Phosphorus is not only an important material basis for plant growth and development,but also an important indicator of soil nutrients.Phosphorus in the soil mainly exists in a poorly soluble state,and a large amount of phosphate fertilizer applied per year is mainly accumulated in an inactive state.Therefore,dissolving and releasing these ineffective phosphorus can increase the effective phosphorus content of the soil,reduce the application of chemical phosphate fertilizer and the consumption of phosphate rock resources.Phosphate solubilizing bacteria（PSB）,one of the soil phosphorus-reducing microorganisms,can convert the ineffective phosphorus in the soil into a plant that can absorb and utilize available phosphorus,and plays an important role in the soil-plant phosphorus cycle system.In this study,a strain of phosphate solubilizing bacteria YL6 isolated from the laboratory and its green fluorescent protein-labeled strain YL6-GFP were selected.Firstly,the optimal growth conditions of YL6 and YL6-GFP were determined,and the biological characteristics of the two strains were compared.Then the chemotaxis test was designed to investigate the chemotaxis of YL6 to carbohydrates and organic acids in root exudates.At the same time,the effect of YL6 on the colonization of Chinese cabbage was studied by exogenous addition of chemotactic agents.Finally,through the combination of pot experiment and soaking seed germination test,the mechanism of YL6 promoting the growth of Chinese cabbage was explored,in order to further understand the interaction between phosphorus bacteria YL6 and Chinese cabbage.The main results are as follows:（1）There was no significant difference in the physiological characteristics of the original strain YL6 and the marker strain YL6-GFP.In the inorganic phosphate-dissolving medium,the optimal culture conditions of YL6 and YL6-GFP are as follows:30°C,pH5.0-6.0,glucose is the suitable carbon source,NH₄NO₃ is the optimal nitrogen source;There was no significant difference in the ability of IAA,GA and EPS,indicating that the introduction of exogenous plasmid GFP did not affect the physiological and biochemical activities of strain YL6.（2）Organic acids can promote the colonization of YL6 in the rhizosphere of Chinese cabbage.The results of beaker method for the determination of YL6-GFP in different growth stages of Chinese cabbage showed that the chemotactic value（che）of YL6-GFP to Chinese cabbage roots was greater than 1,which proved that it had positive chemotaxis to Chinese cabbage roots.,The results of chemotaxis test showed that YL6-GFP has a chemotactic effect on various sugars and organic acids in root exudates,and YL6-GFP had strong chemotaxis to sorbitol and malic acid.Design pot experiment,exogenous single or mixed application of three organic acids（malic acid,citric acid and succinic acid）to the rhizosphere soil of Chinese cabbage can increase the number of colonization of YL6-GFP in root surface and root tissue.The effect of 100mmol/L malic acid was the most significant in single treatment,compared with the control,the colonization of YL6-GFP in the root surface and root tissues of Chinese cabbage was increased by 9.1-fold and 7.5-fold,respectively.In the mixed treatment,the three kinds of organic acids were mixed in the same ratio,and the number of colonization of YL6-GFP in the root surface and root tissues of the Chinese cabbage was increased by 3.4-fold and 4.1-fold compared with the single YL6-GFP treatment.（3）YL6-GFP can colonize and migrate in the root soil and plant tissues of Chinese cabbage.From the vertical level,the colonization and migration order of YL6-GFP in soil is surface layer（0-6cm）→middle layer（6-12cm）→bottom layer（12-18cm）.The number of YL6-GFP colonies recovered and observed by fluorescence microscopy revealed that YL6-GFP appeared in the order of roots→stems（short stems）→petioles（functional leaves→young leaves）→veins→mesophyll cells（functional leaves→Young leaves）.On the roots of Chinese cabbage,YL6-GFP was mainly distributed in root hairs,root epidermal cells and vascular bundles（secondary xylem and phloem）;it is mainly distributed in cortical cells,secondary xylem vessels and myeloid parenchyma cells in the shortening stem;it can be distributed in cortical cells and vascular bundles of the petiole;at the same time,there is YL6-GFP distribution in primary cortical cells,vascular bundles and mesophyll cells.Therefore,this experiment suggests that the phosphate-dissolving bacteria YL6 is likely to migrate from the root to the aerial part through the vascular bundle and the intercellular space.（4）The promoting effect of YL6 on Chinese cabbage was the result of the combination of available phosphorus,IAA and GA.The pot experiment showed that YL6-GFP significantly increased the agronomic traits,biomass,chlorophyll content and plant phosphorus accumulation of Chinese cabbage.Compared with the control,the number of leaves,plant height and root length of Chinese cabbage increased by 33.33%,22.30%and 44.44%,respectively,the fresh weight and dry weight of plants increased by49.90%and 47.59%.the chlorophyll content and phosphorus accumulation of the plants increased by 23.36%and 59.53%.In summary,YL6 can promote the growth of Chinese cabbage.The soaking germination test showed that YL6 fermentation broth could promote the seed germination rate and seedling vigor of Chinese cabbage.Compared with the control,YL6 fermentation broth cultured for 24h had the best effect on seed germination rate and seedling vigor,which increased by 12.5%and 43.6%,respectively.According to the concentration of available phosphorus,IAA and GA in the fermentation broth for 24h,the control experiments of P,IAA and GA solutions were prepared.The results showed that all three substances could increase the seed germination rate and seedling vigor.The three kinds of mixed treatments had the best effect,and the seed germination rate and seedling vigor increased by 16.3%and 33.5%compared with the control.