Effects of nutrient sources on the JK-SH007 growth threshold and IAA secretion
The results for JK-SH007 secreting IAA are shown in Figure 1A. The nutritional conditions were changed to induce JK-SH007 to form a biofilm. The IAA content in the metabolites was 1.1563 µg/mL, which was an increase of 0.2001 µg/mL over that of the planktonic form. IAA is a hormone that promotes plant growth, and changes in the content of metabolized IAA will impact the ability of JK-SH007 to promote growth as a biocontrol agent on poplar. At suitable temperatures, JK-SH007 grows rapidly in a closed medium, and the number of cells reaches a growth threshold when nutrients are depleted. In TSB medium, JK-SH007 grew and metabolized in planktonic mode and grew after 48 h. The cell density reached its highest value, the OD600 value was 2.5559, and the dry cell weight was measured after drying to be 0.035 g; in the treatment group with Mg2+ and glycerol added to TSB medium, JK-SH007 grew and metabolized as a biofilm. After 48 h, the cell density reached the highest value, and the OD600 value was 2.7156. The dry cell weight was 0.192 g, which was 3-10 times higher than that of the planktonic group, and the number of cells was significantly increased (Figures 1B, 2A).
Effects of the biofilm state on the growth of JK-SH007 under different stresses
Temperature stress studies indicate that JK-SH007 in the biofilm group had significant resistance to temperature stress. Under high temperature stress, the OD600 value was 2.66, while the cell concentration of the planktonic bacteria group was only 1.912; under low temperature stress, both groups of bacteria grew poorly, but the OD value of the biofilm group was slightly higher than that of the planktonic group (Figure 2B).
The results of the pH stress studies are shown in Figure 2C. After Mg2+ and glycerin were added, the adaptability of JK-SH007 to an alkaline environment was significantly improved, and the measured OD600 value was 2.773; without these two nutrients, JK-SH007 grew in planktonic mode, and in TSB medium at pH 9, its growth was significantly reduced. The cell concentration was measured after 48 h, and the OD600 value was only 1.859.
At a high concentration of Cu2+, the growth of planktonic JK-SH007 was inhibited. After 48 h of shaking culture, the OD600 value of the fermentation broth was 2.017, which was less than those of the high temperature stress and alkali sexual stress groups, while in the treatment group, even when grown at a Cu2+ concentration as high as 70 mM, JK-SH007 still had high growth. After 48 h of shaking culture, the OD600 of the fermentation broth was 2.694 (Figure 2D).
Determination of salt resistance of JK-SH007 progeny cells
The previous experimental results show that changing the nutrient source during fermentation and inducing JK-SH007 to form a biofilm will increase its potential use in all aspects. The progeny cells from the two forms of JK-SH007 should respond to high concentrations of NaCl. Experimental results showed that the progeny of planktonic JK-SH007 reduced their growth when the NaCl concentration in the fermentation broth reached 2.6 g/100 mL. The growth limit and increasing cell density will cause the cells not to grow. After JK-SH007 is subjected to Mg2+ and glycerol, it grows and metabolizes as a biofilm. Its progeny cells grow in environments without Mg2+ and glycerol, and their strong resistance is retained. When the NaCl concentration reached 4 g/100 mL, the growth of cells began to be inhibited, and the growth of JK-SH007 stopped only when the concentration reached 6 g/100 mL (Figure 3A).
Effect of the biofilm morphology on Populus colonization by JK-SH007
JK-SH007 colonization in poplars can reflect the underlying colonization rules, and both planktonic and biofilm colonization showed a trend of rising first, then falling, and then stabilizing. The cells entered a relatively stable period after 35 days. However, the colonization number for biofilm JK-SH007 was higher than that for planktonic cells. The dynamics of biofilm colonization in poplars are shown in Figure 3B. The relative colonization number on day 7 was 9.08 log10CFU, which slowly decreased to 7.19 log10CFU in 28 days and finally became 8.11 log10CFU after 35 days. The colonization number for poplars with planktonic cells was 8.6427 log10CFU on the 7th day, and it decreased slowly. At 28 days, it reached a minimum value of 7.11 log10 CFU, and then it increased to 7.60 log10 CFU on the 35th day.
Electron microscopy observation after colonization of popular by JK-SH007
Electron microscopy showed that JK-SH007 grows in TSB medium with or without Mg2+ and glycerol. The results are shown in Figure 4. Normally grown JK-SH007 exhibits obvious colony division, and bacteria survive in a single mode of typical planktonic growth (Figure 4A, 4B); JK-SH007 grows on TSB medium supplemented with Mg2+ and glycerol, and the colonies converge together to form an organized three-dimensional community. It was found under an electron microscope at 30,000× magnification that a single bacterial cell appears in a layer of material, which is a typical biofilm structure (Figure 4C, 4D).
We observed JK-SH007 colonization on poplar. Planktonic cells were single bacteria scattered on the leaf tissue of poplar (Figure 5A, 5B), while for biofilm JK-SH007, some bacteria converge together to form cell clusters at 10,000× magnification. The cell clusters have the typical three-dimensional structure of biofilms (Figure 5C, 5D). This morphology can improve the interspecies communication of bacteria and cope with external adverse conditions. It is worth noting that although JK-SH007 gathers in parts of the poplar body, it does not affect the growth of the poplar.
Effect of the biofilm morphology on the compatibility of JK-SH007 and colonized poplar
The metabolites of JK-SH007 were inoculated into poplar seedlings to study the affinity of poplar to JK-SH007. Normally cultured JK-SH007 metabolites were inoculated into poplar tissue culture seedlings. After growing for a period of time, the leaves became green, and the entire plant turned white and eventually died. Although some leaves of the seedlings turned red, they were not lethal, and after a period of adaptation, the growth improved, and the symptoms were alleviated. Through the influence of metabolites on poplar, it is proven that the biofilms have a higher affinity for poplar than the planktonic cells do during inoculation (Figure 6).
JK-SH007 promotes the growth of poplar
In this experiment, we performed a hydroponic test on poplar to examine the correlation between JK-SH007 growth promotion and the cell forms. Figure 7 shows that the both addition and absence of Mg2+ and glycerol in the culture medium led to JK-SH007 having a significant effect on the promotion of poplar germination. In the water control group (Figure 7-A), there was less germination in the front cuttings of poplars and sparse branches and leaves, and some leaves were curled and had an increased mortality rate. The germination effect of the planktonic group was better than that of the control group. The number of plants increased by 75%, the leaf area increased by 64%, and the mortality rate decreased by 10% (Figure 7-B). The shoot growth in the biofilm group was significantly improved compared with the growth in the planktonic group, the number of leafed plants increased by 100%, the leaf area increased by 300%, and the mortality rate decreased by 20% (Figure 7-C). The data show that the performance of biofilms in promoting growth is greater than the performance of planktonic bacteria in promoting growth.