Effects of Biolm Burkholderia Pyrrocinia JK-SH007 On The Colonization And Growth Promotion of Poplar

Burkholderia pyrrocinia JK-SH007 is a high-potential biological control strain. We changed the composition of medium during the fermentation of JK-SH007 cells and induced these cells to form a biolm. In this experiment, we deeply studied the biolm physical and chemical properties. The new fermentation process improves the colonization ability of JK-SH007 and promotes poplar growth. In addition, the biolm bacterial concentration reached 10 10 CFU/mL, the cell dry weight increased over that of a control by 3-10-fold, there was increased environmental stress resistance and IAA secretion, and progeny cells retained resistance to adverse environments. The new biolm cells were applied to poplar. The JK-SH007 colonization ability was improved in the biolm, and some bacteria existed as biolms (cell clusters) in poplar, which would promote forming a dominant niche. Biolm JK-SH007 has an increased anity for poplar during colonization and promotes poplar growth under hydroponic conditions, proving the reliability of the new morphology for treating poplar ulcer disease. This work further provides a theoretical basis for commercially producing JK-SH007.

and changes in bacteria between forms found in nature, planktonic and bio lms (Sauer K et al.2002;Schembri et al. 2003). Although the structure of a single bacterium itself has not changed signi cantly within bio lms, it is combined into a group connected by extracellular polysaccharides and other media.  Barbara et al. 2015). Bio lm is a protection mode of bacteria themselves, especially in complex eld environments. Bio lms are particularly important for the competitive status of species. In terms of biological control, Ji et al. found that the formation of a bio lm by Bacillus is bene cial to controlling bacterial wilt of various host plants (Xianling et al.2008).
Zhao found that β-glucan can promote the formation of Cryptococcus podzolica bio lms and improve the biological control ability of Penicillium pears (YunWang et al. 2018). Dongling et al. found that Pseudomonas putida has a better control effect on tomato bacterial wilt as a bio lm than as planktonic cells (Dongling Sun et al.2017).
Burkholderia pyrrocinia JK-SH007 belongs to the onion Burkholderia genotype IX and is used as a biocontrol agent in the prevention and treatment of poplar ulcer disease in China. According to reports, JK-SH007 is applied as a biofertilizer to the soil environment of poplar roots. It not only improves soil growth and biodiversity and poses little threat to indigenous microorganisms in the environment but also promotes the growth of poplars and increases the resistance of poplars to pathogenic fungi, reducing the occurrence of poplar ulcer disease (Ren et al.2011;Ren et al.2016). In practical applications, due to the stabilization of the environment itself and the application of JK-SH007 in a complex and changing natural environment, the number of poplars that can be successfully colonized reaches the highest value in a short period of time, then decreases with time, and eventually stable low numbers even become zero (Michaela, C.et al.2014). This is because its own phytoplankton morphology results in rapid growth, so the phytoplankton morphology is the most common pattern in the growth history of the bacterium. However, with the continuous coevolution of microorganisms and hosts in this nature, most pathogens have evolved a set of protective modes: bio lms to improve their tolerance and pathogenicity. As a result, when JK-SH007 is commonly used for poplar biocontrol inoculation, repeated inoculations are required over time, which severely restricts the marketization of the bacteria. In this paper, the related functions of JK-SH007 in a bio lm morphology were analyzed by using cell culture, molecular biology, and plant culture. Studying whether adding nutrients to the medium can strengthen bacterial resistance and promote colonization in poplar allows bacteria to be better applied to the prevention and treatment of poplar ulcer disease.

Materials And Method
Microorganism Burkholderia pyrrocinia JK-SH007 was isolated from poplar and was deposited at the Chinese Type Culture Collection (CCTCC: M209028).
The effect of nutrient source on the growth threshold of JK-SH007 The purpose of this experiment was to determine the upper limit of JK-SH007 cell density in the fermentation stage as a bio lm. The control group used TSB medium (15 g/L tryptone, 5 g/L soya peptone, and 5 g/L NaCl), and the experimental group contained two additional nutrients in TSB medium: MgSO 4 7H 2 O and glycerol. The nal concentration of MgSO 4 7H 2 O was 25 mM, and the ratio of glycerol to TSB medium was 1%. For a related scheme, refer to the article by Wiyono et al. ( Wiyono, S et al.2008).
Uncontaminated JK-SH007 glycerol bacteria were removed from a -80 ℃ refrigerator, and single colonies were cultivated on LB solid medium (Gideon et al.2016). A sterile toothpick was used to pick a single colony and immerse it in LB liquid medium. It was activated at 28 ℃ and 180 r/min in a shaker, and the culture was shaken for 24 h to obtain a bacterial suspension.
Then, 20 mL of test medium was added to a 50 mL Erlenmeyer ask, and the bacterial suspension obtained in the early stage was inoculated into the test medium at a concentration of 1%. The culture conditions were 28 ℃ and 180 r/min. The incubation time was 60 h, and three replicates were set for each treatment group.
The growth of JK-SH007 was evaluated by an ultraviolet spectrophotometer: a 48 h bacterial suspension was centrifuged to remove the medium and metabolic products to obtain cells, sterile water was added, and the mixture was stirred to obtain a resuspended solution. Ultraviolet spectrophotometry was used to measure the absorbance of the resuspended solution at 600 nm. According to a growth standard curve, the cell density was obtained.
The resuspended solution was centrifuged again, the supernatant was discarded, the cell pellet was placed in an oven and dried at 70 ℃, and the dry weight of the cells was determined (Hoang-Minh

Determination of resistance to environmental stress in bio lms
The stress resistance of JK-SH007 bio lm cells was explored by changing the medium composition, culture conditions and temperature, alkali, and heavy metal stress conditions in an arti cial culture. An indepth study of bacterial growth and cells under different levels of environmental stress was performed.
The control group used TSB medium as the main component, and the treatment group had Mg 2+ and glycerin added to the TSB medium.
The preparation of the activated bacterial suspension was as described above.
Temperature stress tests: 20 mL of test medium was added to 50 mL Erlenmeyer asks, and the bacterial suspension obtained in the previous stage was inoculated at 1% concentration in the test medium. For low temperature stress, the culture temperature was set to 4 ℃, and the culture was kept in the dark. The absorbance was determined at 600 nm every 24 h for 7 consecutive days; for high temperature stress, the culture temperature was set to 37 ℃, and the shaking speed was set to 180 r/min. After the shaking culture, the absorbance of the bacterial solution at 600 nm was measured after 48 h.
Alkali stress tests: 20 mL of test medium was added to a 50 mL conical ask, the pH of the test medium was adjusted to 9, and the activated bacterial suspension was inoculated at 1%. Since 28 ℃ is a suitable temperature for the growth of JK-SH007, the growth of JK-SH007 in the two forms was almost the same. To reduce the in uence of temperature conditions on the experimental results, this experiment used a cultivation temperature of 28 ℃ and a rotation speed of 180 r/min in a shake culture. Forty-eight hours later, the absorbance of the bacterial solution was measured at 600 nm.
Heavy metal stress tests: 20 mL of test medium was added to a 50 mL conical ask, and CuSO 4 ·5H 2 O was added to the test medium to ensure that the nal concentration was 70 mM. The activated bacterial suspension was inoculated at 1%. In this experiment, the culture temperature was set at 28 ℃, and the rotation speed was 180 r/min. The absorbance of the bacterial solution was measured at 600 nm after 48 h.
Three repetitions were performed for each treatment group.
Effects of nutritional source on the secretion of IAA by JK-SH007 In this experiment, we compared the content of indole-3-acetic acid (IAA) in the metabolites of JK-SH007 in the planktonic and bio lm states. The control group used TSB medium; the treatment group also had Mg 2+ and glycerol in TSB medium to ensure the formation of bio lms. The culture conditions were dark at 37 ℃ and 180 r/min, and the culture time was 48 h. Afterwards, the bacterial suspension was centrifuged to remove the bacteria, and the ability of the two forms to produce IAA was determined by Three repetitions were performed for each treatment group.
Determination of salt resistance of JK-SH007 progeny cells Bio lms of JK-SH007 are produced by the change in fermentation conditions; in actual application, the environment does not have the conditions for bio lm formation. The purpose of this experiment is to study the bacteria in the bio lm morphology, out of the environment, and to determine whether progeny cells retain their stress resistance.
The test medium was prepared as described above, and a suspension of planktonic JK-SH007 cells and a bio lm suspension were obtained. Through centrifugation, the supernatant was discarded, the cells were resuspended, and the OD 600 values of the resuspension solutions were adjusted to be the same.
Three repetitions were performed for each treatment group.
Electron microscopy observation after colonization of poplar with JK-SH007 JK-SH007 morphological observation was performed in the following manner. TSB medium was used to prepare a suspension of JK-SH007 planktonic bacteria. Mg 2+ and glycerin were added to the TSB medium to prepare the JK-SH007 suspension as a bio lm. The bacterial suspension was added to 12well cell culture plates, and a sterile cover glass was placed vertically in each well. They were sealed and allowed to stand for 6 days. After the bio lms matured and stabilized, the coverslip was removed, a scalpel was used to scrape the bacteria, and the bacteria were placed in a phosphate (0.1 M) glutaraldehyde (2.5%) buffer solution and xed. After that, the sample was cleaned with distilled water and ethanol, dehydrated, placed in professional equipment for gold spraying, and nally observed using a eld emission scanning electron microscope.
The colonization of poplar by JK-SH007 as a function of morphology was characterized in the following manner. TSB medium was used to prepare a suspension of JK-SH007 planktonic bacteria, and Mg 2+ and glycerin were added to the TSB medium to prepare a JK-SH007 bio lm suspension. Then, sterile poplar seedlings were inoculated with 2 mL of bacterial suspension. After 7 days, leaf tissue was collected from each sample, and the collected poplar tissue was placed into phosphate (0.1 M) glutaraldehyde (2.5%). In this buffer, electron microscopy observations were performed as above.
Effect of the bio lm morphology on Populus colonization by JK-SH007 TSB medium was used to prepare a bacterial suspension of planktonic JK-SH007 bacteria, and Mg 2+ and glycerin were added to the TSB medium to prepare a bio lm JK-SH007 suspension. A centrifuge and distilled water were used to adjust the OD600 of the bacterial suspension to identical values, sterile poplar seedlings were inoculated with 2 mL of the resuspended solution, and the entire poplar sterile seedlings were extracted at 7, 14, 21, 28, and 35 days, They were weighed and put in a dry pot, and a relative volume of distilled water according to weight was added. They were ground, and tissue uid was collected and poured into a 2 mL centrifuge tube, where it was allowed to stand for 15 minutes. The precipitate was discarded, the tissue uid was diluted to a range of concentrations, and the number of JKsamples were determined by the coating method The relative number of JK-SH007 cells that had colonized poplar was determined.
Effect of the bio lm morphology on the compatibility of JK-SH007 and colonized poplar In this experiment, JK-SH007 colonized poplar as a bio lm, and its metabolic products changed the poplar a nity. Young and sterile healthy seedlings and TSB medium were chosen to prepare fermentation broth with JK-SH007 planktonic bacteria, while Mg 2+ and glycerol were added to TSB medium to prepare fermentation broth for JK-SH007 bio lms. Samples were centrifuged at 10,000 rpm for 2 minutes. The precipitate was discarded, a sterile fermentation broth (containing bacterial metabolites) of JK-SH007 was prepared, and sterile poplar seedlings were inoculated with the metabolic products in excess. The seedlings were placed in a light incubator and grown in a simulated natural environment, and the growth of poplar seedlings was monitored.
Ten repetitions were performed for each treatment group.
JK-SH007 promotes the growth of poplar A fermentation broth was prepared for planktonic JK-SH007 bacteria using TSB medium, while Mg 2+ and glycerin were added to TSB medium to prepare a fermentation broth for JK-SH007 bio lms. The fermentation conditions were 37℃and shaking at 180 r/min in the dark for 48 h.
Annual poplar seedlings were selected, 15 cm cuttings were cut, and cutting seedlings of the same size, individual biomass, tree height and crown width were selected. They were robust and free from diseases and insect pests for hydroponic experiments (Xu et al.2015). The Hoagland nutrient solution for the hydroponic system was chosen, the pH of the hydroponic system was adjusted to 7.0 with NaOH solution, and the volume of each bottle was 300 mL to ensure a constant volume of hydroponic solution. A positive control group was set up (planktonic JK-SH007), a negative control group had distilled water, a treatment group had JK-SH007 bio lm, and there were three other working groups, with each treatment having 10 strains. The nutrient solution was changed every 10 days, and after the nutrient solution was

Result
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 bio lm. 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 OD 600 value was 2.5559, and the dry cell weight was measured after drying to be 0.035 g; in the treatment group with Mg 2+ and glycerol added to TSB medium, JK-SH007 grew and metabolized as a bio lm. After 48 h, the cell density reached the highest value, and the OD 600 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 signi cantly increased ( Figures 1B, 2A).
Effects of the bio lm state on the growth of JK-SH007 under different stresses Temperature stress studies indicate that JK-SH007 in the bio lm group had signi cant resistance to temperature stress. Under high temperature stress, the OD 600 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 bio lm 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 Mg 2+ and glycerin were added, the adaptability of JK-SH007 to an alkaline environment was signi cantly improved, and the measured OD 600 value was 2.773; without these two nutrients, JK-SH007 grew in planktonic mode, and in TSB medium at pH 9, its growth was signi cantly reduced. The cell concentration was measured after 48 h, and the OD 600 value was only 1.859.
At a high concentration of Cu 2+ , the growth of planktonic JK-SH007 was inhibited. After 48 h of shaking culture, the OD 600 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 Cu 2+ 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 bio lm 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 Mg 2+ and glycerol, it grows and metabolizes as a bio lm. Its progeny cells grow in environments without Mg 2+ 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 bio lm morphology on Populus colonization by JK-SH007
JK-SH007 colonization in poplars can re ect the underlying colonization rules, and both planktonic and bio lm colonization showed a trend of rising rst, then falling, and then stabilizing. The cells entered a relatively stable period after 35 days. However, the colonization number for bio lm JK-SH007 was higher than that for planktonic cells. The dynamics of bio lm colonization in poplars are shown in Figure 3B. The relative colonization number on day 7 was 9.08 log 10 CFU, which slowly decreased to 7.19 log 10 CFU in 28 days and nally became 8.11 log 10 CFU after 35 days. The colonization number for poplars with planktonic cells was 8.6427 log 10 CFU 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 Mg 2+ 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 Mg 2+ and glycerol, and the colonies converge together to form an organized three-dimensional community. It was found under an electron microscope at 30,000× magni cation that a single bacterial cell appears in a layer of material, which is a typical bio lm 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 bio lm JK-SH007, some bacteria converge together to form cell clusters at 10,000× magni cation. The cell clusters have the typical three-dimensional structure of bio lms ( 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 bio lm morphology on the compatibility of JK-SH007 and colonized poplar
The metabolites of JK-SH007 were inoculated into poplar seedlings to study the a nity 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 in uence of metabolites on poplar, it is proven that the bio lms have a higher a nity 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 Mg 2+ and glycerol in the culture medium led to JK-SH007 having a signi cant 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 bio lm group was signi cantly 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 bio lms in promoting growth is greater than the performance of planktonic bacteria in promoting growth.

Discussion
We performed relevant functional tests on JK-SH007 bio lms, and the results showed that the population density increased compared to that of planktonic bacteria. We speculate that the formation of bio lms promotes the uptake of nutrients and upregulates the expression of related gene (Michaela et al.2014) resulting in increased cell viability and cell aggregation, which leads to more e cient use of space. The cell growth threshold is increased for bio lm cells. Rosier reported that Pseudomonas aeruginosa increased its cell concentration and diversity of metabolites under the formation of bio lms (Carl L et al.2019). Saheli et al. found that when D-tryptophan (D-TRP) was used to inhibit the formation of Pseudomonas bio lms, the cell adhesion of Pseudomonas decreased, and the cell concentration also decreased signi cantly (Ghosh, S et al.2019). This experiment proves that increasing the concentration of bacteria to promote JK-SH007 bio lm formation without relying on expensive nutrients is feasibly. This provides a new theoretical basis for fermentation conditions for bacterial market application.
According to the results of the stress resistance experiment, bio lms increase JK-SH007 stress resistance to adverse environments. For example, in an environment with a high concentration of heavy metals, the growth of bio lm JK-SH007 was hardly affected. Bio lms are a mechanism of bacterial resistance During the growth of JK-SH007, the composition of the culture medium was changed. We observed differences in cells through an electron microscope and found that under the stimulation of two nutrients, the surfaces of the bacteria were covered with a layer of polysaccharides. There are obvious biological metabolic pathways in the community, which formed a highly stable and organized group living together. This explains the formation of bio lms, and Gao et al. observed the same results using an electron microscope (Tantan Gao et al.2015). When JK-SH007 was applied as a bio lm to sterile poplar, a cell cluster structure was found on the leaves. Generally, the formation of cell clusters produces a large amount of metabolites in a certain part of the host body, which will not be conducive to the host's growth. For bio lm JK-SH007, it may be that the cells are wrapped by exosporium, which causes regular growth and metabolism among species (Abdelkarim et al.2017). As a result, the impact on the poplar itself becomes low. Through an analysis of the colonization number at different times, the colonization number of bio lm JK-SH007 inoculated in poplar trees was higher than that of planktonic bacteria in each time period. In the wild, high-density colonization of biocontrol strains in the surrounding environment of the host can effectively compete with other pathogens for space and nutrients, which is considered to be an important biocontrol mechanism (Jia Liu et al.2013). In addition, the a nity experiment con rmed that even if a group of cells gathered together for growth and metabolism, it did not have an adverse effect on the poplar, and due to changes in metabolites, it actually increased the a nity for the poplar. This indicates that JK-SH007 adopting the bio lm morphology increases its colonization ability and a nity for the host, which will help it occupy a dominant niche in the complex microbial community around the host. In a similar study, some scholars used Paraburkholderia tropica to colonize barley to increase grain production (García et  . JK-SH007, as a high-potential biocontrol bacterium, grows under normal conditions, and it has di culty forming bio lm. The reason may be that it has a high expression of movement-related genes during the growth stage, and it has di culty clustering. When applied in the wild environment, planktonic JK-SH007 has much lower vitality than the bio lm JK-SH007, which will affect its biological control effect. This study has a good result for this problem. This experiment provided the possibility that bio lm JK-SH007 could be used as a biological agent in the eld. IAA is a hormone used to stimulate plant growth. It can promote the growth and development of plants within a certain concentration range. IAA produced by the metabolism of bene cial bacteria generally promotes the growth of plant organs (Li Xu et al.2015). JK-SH007, as a bene cial antibacterial, can also secrete IAA itself (Wan-Hui, Liu et al.2019). We con rmed through experiments that as a bio lm, its metabolic IAA content increased. The subsequent growth-promoting poplar hydroponic experiments also con rmed that JK-SH007 cultured with Mg 2+ and glycerin in the nutrient source promoted the germination and growth of plants. The disadvantage is that this experiment was conducted under hydroponic conditions, which is more conducive to the metabolism of bacteria than soil is, i.e., in the wild environment. Can JK-SH007 have the same growth promotion in the soil environment? The effect will be the focus of future research.
These experimental results show the importance of adding nutrients during the fermentation of JK-SH007 and its possible use to control poplar ulcer disease. The addition of nutrients can change the morphology of JK-SH007 bio lms, and these experiments proved the advantages of bio lms in terms of stress resistance, a nity, and growth promotion. In China, poplar is used as a shelterbelt for large-scale planting, and a single variety causes a large-scale outbreak of poplar ulcer disease during drought (Chen et al.2012). The application of JK-SH007 as a biological fertilizer in the eld has become an important topic. This study is the rst report that adding nutrients during the fermentation stage changes its metabolism to make it more effective in performing related functions around the poplar. When JK-SH007 is commercialized as a biological fertilizer, this study can be used as an important reference. In follow-up research, the potential biological control ability of bio lm JK-SH007 after poplar colonization can be considered.

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Ethics approval and consent to participate All the authors listed have approved the present submitted version. And does not involve ethical issues.

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Competing interests
I would like to declare on behalf of my co-authors that any gure or text taken from another paper is clearly indicated with the full source and permission of the authors of said source. Besides, our manuscript has not been published previously, and not under consideration for publication elsewhere, in whole or in part.   Effects of JK-SH007 metabolites on poplar Note: The left picture shows the growth of poplar seedlings inoculated with planktonic JK-SH007 metabolic uid; the right picture shows the growth of poplar seedlings inoculated with metabolic uid from bio lm JK-SH007.