CN114517166B - Application of fructose-1, 6-calcium diphosphate in improving plant rhizosphere colonisation of growth-promoting bacteria - Google Patents
Application of fructose-1, 6-calcium diphosphate in improving plant rhizosphere colonisation of growth-promoting bacteria Download PDFInfo
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Abstract
The invention discloses an application of a high-energy phosphoric acid compound fructose-1, 6-diphosphate calcium salt (FB) in improving the colonization of growth-promoting bacteria on the rhizosphere and preparing a preparation of the growth-promoting bacteria on the colonization of plant rhizosphere. The signal molecule FB for promoting the colonization of the growth-promoting bacteria at the rhizosphere is obtained through screening, and the FB is found to obviously increase the chemotactic effect, the group swimming effect and the biofilm formation of the plant rhizosphere growth-promoting bacteria, so that the colonization of the growth-promoting bacteria at the plant rhizosphere is obviously increased.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of fructose-1, 6-diphosphate calcium salt in improving colonization of plant rhizosphere by growth-promoting rhizobacteria.
Background
Due to the overapplication of chemical fertilizers in agricultural production, a series of problems of ecological safety, food safety, environmental pollution and the like are caused. Microbial fertilizers are widely paid attention to because of the characteristics of green environmental protection, safety, effectiveness and the like, and become a novel driving force for the development of green ecological agriculture. For 20 years, the microbial fertilizer industry in China has undergone a high-speed development stage. At present, the development of new products, new strains, new processes, new effects and the like in the period of most innovation is a development target of the industry in the new period.
The plant rhizosphere growth promoting bacteria (Plant Growth Promoting Rhizobacteria, PGPR) are beneficial bacteria living in rhizosphere soil or attached to plant root systems, and can not only provide nutrition for plants and reduce the occurrence of plant diseases and insect pests, increase crop yield, but also improve soil quality and prevent secondary pollution. Currently, PGPR has become one of the hot spots in microbial fertilizer research. The effect of rhizosphere growth-promoting bacteria in promoting plant growth and improving soil health depends on the processes of colonization of plant rhizosphere, induction of plant system resistance, secretion of specific biocontrol active substances and the like.
The biological film has important functions in the processes of adapting plant rhizosphere growth promoting bacteria to environmental stress, improving the sensitivity of pesticides and the like, effectively promoting crop growth and the like. It has been found that in primary metabolism, saccharides secreted by the root system of Arabidopsis can attract Bacillus subtilis, and malic acid secreted by the root system can recruit Bacillus subtilis to the root system and stimulate it to form a biofilm. A variety of L-amino acids have chemotactic effects on P.fluorescens. During secondary metabolism, flavonoids from wheat root secretions stimulate chemotactic reactions by sessile sesbania. The research shows that the chemotaxis of the plant to root secretions of different plants is different, and the chemotaxis and research effects of different subspecies strains to the plant are also different. Therefore, finding signal molecules for rhizosphere colonization for high-efficiency azotobacter is a bottleneck for restricting popularization and application.
In the prior art, the high-energy phosphoric acid compound fructose-1, 6-diphosphate calcium salt (FB) is utilized, and the chemical formula C 6 H 12 CaO 12 P 2 CAS registry No. 103213-33-8 is used in medicine for the adjuvant treatment of myocardial ischemia. No study on the enhancement of plant rhizosphere colonization by the growth-promoting bacteria serving as a signal molecule is reported.
Disclosure of Invention
The invention provides an application of a high-energy phosphoric acid compound fructose-1, 6-diphosphate calcium salt as a signal molecule for improving growth-promoting bacteria in plant rhizosphere colonisation.
Wherein, the fructose-1, 6-calcium diphosphate improves the formation amount of biological film and the swimming diameter of the growth-promoting bacteria.
Specifically, the growth-promoting bacteria are plant rhizosphere growth-promoting bacteria, including but not limited to any one of Bacillus subtilis (Bacillus subtilis) NRCB002, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) NRCB005, pseudomonas stutzeri (Pseudomonas stutzeri) NRCB010, bacillus albus (Bacillus albus) Lv5A and Achromobacter denitrificans (Achromobacter denitrificans) YSQ 030.
Wherein, bacillus subtilis (Bacillus subtilis) NRCB002 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.17213 and the preservation address of national institute of sciences of China, national academy of sciences of China, national institute of sciences of China, no. 3, beijing area, and the detailed information of which is disclosed in patent CN 201910191640.8. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) NRCB005 was deposited at China general microbiological culture Collection center, china Committee for culture Collection of microorganisms, with a accession number CGMCC No.17214, and detailed information thereof is disclosed in CN201910191639.5, 1 and 18. Pseudomonas stutzeri (Pseudomonas stutzeri) NRCB010 was deposited at China general microbiological culture Collection center, with the accession number CGMCC No.19067, and detailed information thereof is disclosed in CN202010071098.5, at 12.02 of 2019. Bacillus albus (Bacillus albus) Lv5A was deposited at the China general microbiological culture Collection center, with the accession number CGMCC No.21616, at the national institute of microbiology, national institute of sciences, 1, 3, beijing, and its detailed information is disclosed in patent CN 113046261A. Achromobacter denitrificans (Achromobacter denitrificans) YSQ030 was deposited in China general microbiological culture Collection center (CGMCC) at 7.8 of 2021, and the deposited address is China center for type 3 microbiological study (China academy of sciences) in the Korean area of Beijing as CGMCC No. 22850.
In some specific embodiments, the present application demonstrates that fructose-1, 6-bisphosphate calcium salt can enhance rhizosphere colonization of tomato, lettuce or rice by rhizosphere growth-promoting bacteria.
The invention also provides application of the fructose-1, 6-diphosphate calcium salt in preparation of a preparation for improving the colonization of plant rhizosphere by the rhizosphere growth-promoting bacteria.
In addition, the invention provides a method for improving the colonization of the rhizosphere growth-promoting bacteria in the plant rhizosphere, and fructose-1, 6-calcium diphosphate is added into the fermentation liquor of the plant rhizosphere growth-promoting bacteria.
Preferably, the concentration of fructose-1, 6-bisphosphate calcium salt is added to the plant rhizosphere growth promoting bacteria fermentation broth to be 0.01% -10%. More preferably, the concentration of fructose-1, 6-bisphosphate calcium salt is added to the plant rhizosphere growth promoting bacteria fermentation broth in the range of 0.1% to 1%.
The invention also provides another method for improving the colonization of the rhizosphere growth-promoting bacteria in the plant rhizosphere, which comprises the step of directly adding fructose-1, 6-calcium diphosphate to the plant rhizosphere, wherein the concentration of the added fructose-1, 6-calcium diphosphate is 0.01-10%. Preferably, the optimum concentration of fructose-1, 6-bisphosphate calcium salt added to the rhizosphere of plants is between 0.05% and 0.2%. The purpose of improving the colonization of the rhizosphere growth-promoting bacteria in the plant rhizosphere can be achieved by adding the fructose-1, 6-calcium diphosphate into the plant rhizosphere or growth-promoting bacteria product.
The beneficial effects are that: the invention provides an application of a high-energy phosphoric acid compound fructose-1, 6-diphosphate calcium salt (FB) in improving the colonization of growth-promoting bacteria on the rhizosphere and preparing a preparation of the growth-promoting bacteria on the colonization of plant rhizosphere. According to the invention, the signal molecule FB for promoting the colonization of the growth-promoting bacteria in the rhizosphere is screened out from the plant rhizosphere, and researches show that the FB has obvious chemotactic effect and group-swimming effect on the growth-promoting bacteria, and can obviously induce the colonization of the growth-promoting bacteria in the plant rhizosphere. The quantity of the growth promoting bacteria of the plant root containing the FB in the rhizosphere secretion is obviously increased, and the FB can effectively promote the formation of the growth promoting bacteria biomembrane. Therefore, the FB has wide application prospect in promoting the growth-promoting bacteria to colonize the rhizosphere.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of FB on the formation of NRCB002 biofilm;
FIG. 2 is a graph showing the effect of different concentrations of FB on the formation of NRCB005 biofilm;
FIG. 3 is the effect of different concentrations of FB on the formation of NRCB010 biofilm;
FIG. 4 is the effect of different concentrations of FB on Lv5A biofilm formation;
FIG. 5 is a graph of the effect of FB on different bacterial surges;
FIG. 6 shows the effect of FB on colonization of tomato rhizosphere by the growth-promoting rhizosphere bacterium NRCB002 (A) the number of viable NRCB002 bacteria colonized by tomato roots under FB treatment (. Times.10) 7 ) The method comprises the steps of carrying out a first treatment on the surface of the (B) Number of active NRCB002 bacteria (10) colonized by tomato root without FB treatment 7 )；
FIG. 7 shows the number of active NRCB002 bacteria (. Times.10) colonizing tomato roots at various concentrations of FB treatment 7 )；
FIG. 8 shows the number of active NRCB002 bacteria (. Times.10) colonized by different plant roots under FB treatment 7 )。
Detailed Description
The present invention will be described in further detail with reference to specific examples. Detailed embodiments and specific operations are given, examples will aid in understanding the present invention, but the scope of the present invention is not limited to the following examples.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 effect of fructose-1, 6-bisphosphate calcium salt (FB) on the formation of biofilms by rhizosphere growth-promoting bacteria NRCB002, NRCB005, NRCB010 and Lv 5A.
Experimental methods
Each strain was inoculated from the plate into a 50ml centrifuge tube, cultured at 30℃and 200rpm for 12 hours, and 1% of the inoculum was aspirated into a 250-flask containing 50ml of LB medium, and cultured overnight.
The bacterial suspension was aspirated into 20ml tubes and centrifuged at 5000rpm for 5min (50 ml centrifuge tube). The cells were collected by washing with physiological saline twice, and the volume was fixed with a K-medium until the OD was about 1.2. To 96-well plastic culture plates, 37.5. Mu.L of bacterial liquid and 112.5. Mu.L of K-type culture medium with different FB concentrations (0, 0.1, 0.5 and 1 g/L) were sequentially added, 8 parallel plates were arranged for each treatment, and the same amount of sterile culture medium was used as a blank group for stationary culture at 28℃for 48 hours.
Culturing for 48h, measuring the absorbance of the solution at OD600nm by using an enzyme-labeled instrument, gently sucking the liquid in the culture plate by using a pipette, and adding 160 mu L of distilled water to clean planktonic bacteria. 160. Mu.L of 2% crystal violet was added to the culture well, the well was stained at room temperature for 10 minutes, the stained solution was aspirated, unbound crystal violet was removed with distilled water, and after air-drying, 160. Mu.L of 30% acetic acid was added to the culture well, and the absorbance of the solution at OD560nm was measured with an enzyme-labeled instrument. Biofilm formation was expressed as absorbance at OD560 nm.
(II) results of experiments
The effect of different concentrations of FB on the formation of the rhizosphere growth-promoting bacteria biofilm is shown in FIGS. 1-4, and tables 1 and 2, and it can be seen that FB can promote the formation of rhizosphere growth-promoting bacteria NRCB002, NRCB005, NRCB010 and Lv5A biofilms; wherein, the forming amount of the rhizosphere growth promoting bacteria NRCB010 biological film is the highest, and the forming amount of the biological film is increased by 256.0 percent, 262.2 percent and 449.8 percent under different concentrations (0, 0.1, 0.5 and 1 g/L) compared with the biological film of a control group; the formation amount of the NRCB002 biological film is respectively increased by 185.2 percent, 192.9 percent and 213.2 percent compared with the control group; the formation amount of NRCB005 biofilm was increased by 22.2%, 24.4% and 67.6% respectively compared with the control group; the Lv5A biofilm formation was increased by 33.06%, 85.83% and 265.96% respectively compared to the control group. The result shows that the FB can effectively promote the formation of the biological film of the rhizosphere growth-promoting bacteria, and is beneficial to improving the colonization of the rhizosphere growth-promoting bacteria in the plant rhizosphere.
TABLE 1
TABLE 2
Example 2 effect of fructose-1, 6-bisphosphate calcium salt (FB) on the swimming diameter of the rhizosphere growth-promoting bacteria NRCB002, NRCB005 and NRCB 010.
Experimental methods
Swimming medium: each 1L contains 10g of tryptone, 10g of sodium chloride, 5g of yeast powder and 5g of agar powder.
Overnight activated strains NRCB002, NRCB005, NRCB010 seed fluid were inoculated using NBNS broth. Culturing at 30 ℃ and 200rpm to mid-log phase (od600=1.0). 100 μl of 0.1g/L FB solution was added to Swiming plates and spread evenly. A round sterile paper sheet with the diameter of 0.5cm is placed in the center of a flat plate, 5 mu l of bacterial liquid is sucked by a gun head and slowly dripped to the center of the paper sheet, the paper sheet is placed still for ten minutes in a sterile environment, a culture medium is gently moved to a 30 ℃ biochemical incubator for culture, and the diameters of bacterial rings formed by NRCB002, NRCB005 and NRCB010 are respectively measured at 12h, 8h and 24h time points. Each group was run in 3 replicates.
As shown in fig. 5, after FB treatment, NRCB002 after 12 hours of cultivation on the surface of the mobility plate, the diffusion diameter was increased by 40.4% compared to the control group; after NRCB005 is cultured on the surface of the swimming flat plate for 8 hours, compared with a control group, the diffusion diameter is improved by 30.1 percent; after NRCB010 is cultured on the surface of the swimming flat plate for 24 hours, compared with a control group, the diffusion diameter is obviously improved by 70.5 percent.
Example 3 FB effect on plant rhizosphere colonization by rhizosphere growth promoting bacteria.
The experiment selects a rhizosphere growth-promoting bacterium NRCB002, and the effect of FB on the rhizosphere growth-promoting bacterium NRCB002 in plant rhizosphere colonisation is studied, and the experimental method is as follows:
tomato seed treatment: treating with 84 disinfectant solution 2.5% for 10min (shaking vigorously), washing with sterile water for 7-10 times, soaking with sterile water for 5min for 2 times, removing water, spreading the seeds on agar plate, and germinating in an illumination incubator.
And (3) strain treatment: the above bacteria were inoculated into 50ml centrifuge tubes containing 10ml of LB medium, cultured at 200rpm and 30℃overnight, and OD600 was adjusted to 1.0, and dilution factors were recorded. The bacterial solution was centrifuged at 5000rpm for 10min, the supernatant was removed, and after 2 times of resuspension with sterile water in an equivalent amount under aseptic conditions, it was diluted 100-fold with sterile water. When the microbial fertilizer or pesticide is used, the microbial fertilizer or pesticide can be uniformly mixed and directly irrigated to the root of a plant.
Culturing plants: (1) soaking tomato seedlings for 3 days in a bacterial liquid for 30min, 60min and 90min under a sterile environment. Subsequently, the treated seedlings were removed, the surface water was blotted with sterile filter paper, and transferred to a 1/10MS solid medium with a FB concentration of 0.05g/L and 0.1 g/L. And (3) parallelly placing seedlings at about 1/3 of the flat plate, wherein seven seedlings are placed in each culture medium, and the directions of the seedlings and the intervals among the seedlings are consistent. The control was 1/10MS medium without FB and repeated 3 times. Transferring to a light incubator for culturing for 7 days, and taking out.
(2) Under the aseptic environment, tomato seedlings which are sprouted for 3 days are soaked in bacterial liquid for 30min. Subsequently, the treated seedlings were taken out, the surface water was sucked up with sterile filter paper, and transferred to 1/10MS solid medium with FB concentration of 0.05g/L, 0.1g/L, 0.2g/L, respectively. And (3) parallelly placing seedlings at about 1/3 of the position of the flat plate, and ensuring that the directions of the seedlings and the intervals among the seedlings are consistent for 7 plants in each culture medium. The control was 1/10MS medium without FB and repeated 3 times. Transferring to a light incubator for culturing for 7 days, and taking out.
(3) Under the aseptic environment, tomato seedlings which are sprouted for 3 days are soaked in bacterial liquid for 30min. Subsequently, the treated seedlings were removed, the surface was blotted with sterile filter paper and transferred to 1/10MS solid medium. Tomato, lettuce and rice seedlings are placed in parallel at about 1/3 of the flat plate, 7 plants are placed in each culture medium, and the seedling direction and the seedling interval are consistent. The control was 1/10MS medium without FB and repeated 3 times. Transferring to a light incubator for culturing for 7 days, and taking out.
Counting: after the tomato seeds are cultivated for 7 days by illumination, the tomato roots are cut off under the aseptic environment and the bacteria with the unclarified surface and the residual culture medium are repeatedly washed by aseptic water. Then, the root is weighed and ground by adding sterile water, and finally the obtained root turbid liquid is diluted to 10 in turn by normal saline -3 、10 -4 、10 -5 100 μl was pipetted onto LB medium and each treatment was repeated 3 times and incubated in a 30℃incubator for 12h. The number of viable bacteria colonized to the tomato root was determined by plate counting. Saline and sterile water from the last alfalfa root rinse were used as negative controls. The count results were normalized by root weight. Colonizing tomatoesThe mass effective viable count of the root is calculated by the following formula:
n (number/g) =x×k×v1/m×v2
Wherein x is the number (number) of living colonies; k is dilution multiple; v1 is total volume (ml) of root turbid liquid; v2 is the addition amount (ml) of the root turbid liquid; m is root mass (g). In order to more intuitively embody the number of effective viable bacteria, log can be used 10 CFU g -1 To express the final measurement result.
As shown in FIG. 6, tomato seedlings are soaked in bacterial liquid for 30min, 60min and 90min and then placed in 1/10MS solid culture medium with FB concentration of 0.1g/L, and the effective viable count for root colonization at different times is 1.58×10 respectively 7 cfu/g、1.71×10 7 cfu/g and 1.65X10 7 cfu/g, while the number of viable bacteria for rhizosphere colonization of the blank group without FB treatment was 5.47×10 6 cfu/g、5.76×10 6 cfu/g and 5.68X10 6 cfu/g, the number of colonisation was increased by 188.4%, 196.9% and 190.5% respectively. The results show that: (1) the number of effective viable bacteria of tomato seedlings soaked for 30min, 60min and 90min is not significantly different in the FB and BLK culture medium application, namely the rhizosphere colonization capability of the strain is not greatly changed within 30-90 min; (2) while strain NRCB002 showed a significant increase in rhizosphere colonization ability after FB application. As shown in FIG. 7, tomato seedlings were immersed in the bacterial liquid for 30min and uniformly placed in 1/10MS solid medium with FB concentration of 0.05g/L, 0.1g/L, 0.2g/L, respectively. The colonization effect of FB concentration of 0.1g/L is best, and the effective viable count reaches 1.63 multiplied by 10 7 The cfu/g and the FB of 0.05g/L and 0.2g/L can enhance the colonization effect of bacteria, and the number of effective viable bacteria for colonization is 7.3X10 respectively 6 cfu/g and 1.18X10 7 cfu/g, number of viable bacteria of rhizosphere colonization of blank group without FB treatment was 4.93×10 6 cfu/g. The results show that: different concentrations of FB can enhance the rhizosphere colonization effect of bacteria, and the effect is best at 0.1 g/L.
As shown in FIG. 8, after the strain NRCB002 is treated by FB, the effective viable count of the rhizosphere colonization of tomato, lettuce and rice seedlings soaked in the bacterial liquid for 30min is 1.39X10 respectively 7 cfu/g、1.65×10 7 cfu/g、9.2×10 6 cfu/g, not appliedThe number of viable bacteria colonized by rhizosphere of the blank group treated by the FB is 5.65X10 6 cfu/g、1.13×10 7 cfu/g、4.77×10 6 cfu/g. Namely after FB treatment, the bacterial strain NRCB002 has enhanced colonization effect on different plants, and the number of effective living bacteria for colonization is respectively increased by 146.0%, 46.0% and 92.9%. The result shows that after FB is added, the strain can promote the colonization effect of the tomato rhizosphere more obviously.
The invention provides a thought and a method for promoting rhizosphere growth-promoting bacteria to colonize plant rhizosphere, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made to those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (6)
1. The application of the fructose-1, 6-diphosphate calcium salt in improving the rhizosphere growth-promoting bacteria in plant rhizosphere colonisation, wherein the fructose-1, 6-diphosphate calcium salt improves the biomembrane formation amount and the migration diameter of the plant rhizosphere growth-promoting bacteria; the rhizosphere growth-promoting bacteria are bacillus subtilis @Bacillus subtilis) NRCB002, bacillus amyloliquefaciens @Bacillus amyloliquefaciens) NRCB005, pseudomonas stutzeri ]Pseudomonas stutzeri) NRCB010, bacillus albus @Bacillus albus) Any one or a combination of a plurality of Lv 5A.
2. The use according to claim 1, wherein the plants are tomato, lettuce and rice.
3. Application of fructose-1, 6-calcium diphosphate in preparation of preparation for improving rhizosphere growth-promoting bacteria in plant rhizosphere colonisation, wherein the rhizosphere growth-promoting bacteria are bacillus subtilis @Bacillus subtilis) NRCB002, bacillus amyloliquefaciens @Bacillus amyloliquefaciens) NRCB005, pseudomonas stutzeri ]Pseudomonas stutzeri) NRCB010, bacillus albus @Bacillus albus) Any one or a combination of a plurality of Lv 5A.
4. A method for improving colonization of rhizosphere growth-promoting bacteria in plant rhizosphere is characterized by adding fructose-1, 6-calcium diphosphate into fermentation liquor of plant rhizosphere growth-promoting bacteria, wherein the fermentation liquor of plant rhizosphere growth-promoting bacteria contains bacillus subtilisBacillus subtilis) NRCB002, bacillus amyloliquefaciens @Bacillus amyloliquefaciens) NRCB005, pseudomonas stutzeri ]Pseudomonas stutzeri) NRCB010, bacillus albus @Bacillus albus) A fermentation broth of any one or a combination of Lv 5A.
5. The use according to claim 4, wherein the concentration of fructose-1, 6-bisphosphate calcium salt added to the plant rhizosphere growth promoting bacteria fermentation broth is 0.01% -10%.
6. The use according to claim 5, wherein the concentration of fructose-1, 6-bisphosphate calcium salt is added to the plant rhizosphere growth promoting bacteria fermentation broth in the range of 0.1% to 1%.
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