CN112662590B - Biocontrol plant endophytic bacterium ZN-S4 and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and discloses Burkholderia gladioli ZN-S4 with the preservation number of CGMCC NO:18963. the invention also discloses the application of the Burkholderia gladioli ZN-S4: the absorption and utilization of the nutrient elements N and P in the soil by the crops are promoted; promoting crop growth or increasing biomass.

Description

Biocontrol plant endophytic bacterium ZN-S4 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an endophytic bacterium burkholderia gladioli strain and an influence effect thereof on plant growth promotion and the like.

Background

The endophytic bacteria of the plant are widely present in tissues such as roots, stems and the like, are not influenced by the external environment, and form a symbiotic relationship with the plant. The bacteria have wide biological effects on the growth and development of host plants, pest and disease damage resistance and the like, and have important significance in the popularization and application of some plants and crops. With the continuous damage to the environment and the destructive utilization of resources by human beings, the ecology continuously deteriorates, the plant resources are subjected to increasingly severe tests, and the influence of the endogenous bacteria on the plants is multifaceted and becomes one of the hot problems of the current domestic and foreign researches. In recent years, beneficial endophytic bacteria and endophytic fungi are found in plants, and the control effect of endogenous strains on plant diseases and biological development and application are attracting much attention of scientists. The endophytic bacteria are mainly distributed in seeds, flowers, stems, leaves and root systems of plants, are in reciprocal symbiosis with the plants, can provide photosynthetic products and minerals, and can stimulate the growth and development of host plants and improve the biotic stress and abiotic stress resistance capability of the host plants, namely, the endophytic bacteria and the host plants have material and energy cyclic communication. The positive growth-promoting effect of the endophytic bacteria on the host plant is partly a result of the nitrogen fixation or secreted active substances of the endophytic bacteria and their secondary metabolites on the growth of the plant.

The prior Burkholderia gladioli and the application thereof comprise:

1. 2015103159781, an invention of "a strain of wild rice endophytic bacteria gladiolus burkholderia Fse32 and application thereof", informs that: burkholderia gladioli (Burkholderia gladioli) Fse32 with a preservation number of: CCTCC NO: M2014672; the strain has the effects of secreting auxin and the like for promoting plant growth and antagonizing the activity of various plant pathogenic bacteria.

2. 2020109948984, the invention of "a strain of litchi endophytic Burkholderia gladioli and its application in preventing and treating anthracnose and frost blight of litchi" teaches that: burkholderia gladioli SZPT16 with the accession number of GDMCC No:61168 has strong inhibiting effect on colletotrichum litchii and peronophythora litchii

3. 2020107574144 of the invention, burkholderia gladioli Nees of dendrobe and its application, teaches Burkholderia gladioli Nees BL-HTie-5 with a preservation number of CGMCC No.18775. Has excellent bacteriostatic activity on both dendrobium pathogenic fungi of the tuber tara (Athellia rolfsii) and the humicola (Myrothecium roridum).

4. 2013103011959, an invention of bacterium having a growth promoting effect in cooperation with AM fungus and application thereof in vegetable growth promotion, informs Burkholderia gladioli (Burkholderiagladioli) CGMCC No.6492 of gladiolus gladioli, and has an outstanding growth promoting effect on vegetables such as cucumber, tomato, hot pepper and eggplant.

5. 201611001344X discloses a Burkholderia bacterium NJAU-B8 with phosphate solubilizing capability and a microbial fertilizer prepared from the same, and the fact that the Burkholderia bacterium NJAU-B8 with phosphate solubilizing capability has strong inorganic phosphate solubilizing capability; can effectively promote the growth of the potted corn plants and increase the field yield of corn, chinese cabbage and potato.

Disclosure of Invention

The invention aims to solve the technical problem of providing burkholderia gladioli ZN-S4 and application thereof, wherein the strain can promote the growth of plants, especially cucumber, tomato and rice.

In order to solve the technical problems, the invention provides Burkholderia gladioli ZN-S4 with the preservation number of CGMCC NO:18963.

the invention also improves the application of the Burkholderia gladioli ZN-S4:

promoting the absorption and utilization of nutrient elements in soil by crops; the nutrient elements are N and P.

Used for promoting the growth of crops or increasing biomass.

Colonizing the roots of the crops, fixing N element in the air, promoting the dissolution of P in soil, promoting the growth of the crops and improving the disease resistance of the crops.

The crops are cucumber, tomato and rice.

The preservation information of the strain ZN-S4 is as follows:

the preservation name is Burkholderia gladioli, preservation unit: china general microbiological culture Collection center, the preservation Address: xilu No.1 Hospital No. 3, kcgmcc NO:18963, time of deposit 2019, 11/18.

The Burkholderia gladiolis ZN-S4 has the following characteristics: the suitable growth temperature of the strain on the NB culture medium is 23 ℃; the bacterial colony is light yellow and round, is wet, sticky and convex, has a smooth surface and regular edges, and has a short rod-shaped thallus and a negative gram stain; the growth is faster. Yielding a water-soluble yellow pigment.

The strain of the invention is isolated from the plant dendrobium officinale belonging to the kingdom of Bacteria (Bacteria), proteobacteria (Proteobacteria), pseudomonas (Pseudomonas), burkholderia (Burkholderia).

Compared with the existing Burkholderia gladiolis ZN-S4, the Burkholderia gladiolis ZN-S4 has the following technical advantages: not only has the growth promoting effect, but also can colonize the roots of plants, fix nitrogen elements in the air, promote the dissolution of P in soil and inhibit the growth of pathogenic bacteria such as rice bacterial leaf blight and the like.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a morphological feature diagram of colony and thallus of Burkholderia gladioli ZN-S4;

in FIG. 1, A is a plate diagram of a colony, B is a front diagram of a single colony, and C is a gram stain diagram of bacteria;

FIG. 2 is a 16S rRNA phylogenetic tree of ZN-S4 strain;

FIG. 3 shows the dry weight growth rate of each variety of rice seedlings at 30 days after seed soaking treatment of ZN-S4 bacterial solutions with different concentrations;

FIG. 4 shows the dry weight increase rate of rice roots of each variety on the 30 th day after the seed soaking treatment of ZN-S4 bacterial solutions with different concentrations;

FIG. 5 shows the effect of ZN-S4 seed soaking on the root length of different varieties of rice;

FIG. 6 shows the effect of ZN-S4 seed soaking on the seedling height of different varieties of rice;

FIG. 7 shows the shape and growth of tomato seedlings after ZN-S4 root irrigation for 21 d;

in fig. 7:

blank control, E: tomato seedlings to which ZN-S4 bacterial liquid is applied, F: tomato seedlings to which ZN-S4 bacterial liquid 100 Xis applied, G: applying ZN-S4 bacterial liquid 200 Xthe tomato seedlings;

FIG. 8 shows the effect of ZN-S4 fungus root irrigation on tomato biomass;

FIG. 9 shows the effect of ZN-S4 bacteria foliar spray on tomato biomass;

FIG. 10 shows the shape and growth of cucumber seedlings after ZN-S4 bacterial liquid is sprayed for 21 days;

in the context of figure 10 of the drawings,

a: control cucumber seedlings without added bacterial liquid, F: and (3) applying ZN-S4 bacterial liquid with the volume of 50 Xthe cucumber seedling, G: and (3) applying ZN-S4 bacterial liquid with the volume of 100 times to cucumber seedlings, H: applying ZN-S4 bacterial liquid with the size of 200 Xthe cucumber seedling;

FIG. 11 shows the influence of ZN-S4 bacterial liquid spray treatment on cucumber biomass;

FIG. 12 shows the influence of ZN-S4 bacterial liquid root irrigation on cucumber biomass;

FIG. 13 shows the amount of ZN-S4 colonized in different tissues of rice;

FIG. 14 shows the colonization dynamics of ZN-S4 mutant strain at root, stem and leaf of rice seedling;

FIG. 15 shows the nifH gene amplification of the endophytic bacteria of Dendrobium officinale Kimura et Migo (ZN-S2 CGMCC NO.18962 in the figure, without nitrogen fixation);

FIG. 16 shows the results of measurement of the effective phosphorus concentration in soil.

Detailed Description

Example 1 isolation, culture and characterization of Burkholderia gladiolis (Burkholderia gladiolis) strains:

beef extract peptone medium (NA): adding distilled water into 5g of tryptone, 3g of beef extract, 1g of yeast powder, 10g of D-glucose agar and 15g of agar to reach the constant volume of 1000mL; conventional autoclaving (1.1 atm, 121 ℃ C. For 20 min) was then carried out. The NB medium was obtained without agar.

The burkholderia gladioli can be obtained by separation culture in a laboratory according to the following conditions.

Collecting and cultivating Dendrobium officinale from Zhejiang, rinsing collected Dendrobium officinale with tap water, sterilizing the Dendrobium officinale rootstock in 75% (v/v) alcohol solution for 30s, then sterilizing in 1% (v/v) sodium hypochlorite solution for 10min, and rinsing with sterile water for 3 times. Cutting root and stem with sterile scissors, grinding with sterile mortar, suspending plant powder with appropriate amount of sterile water, dipping suspension with inoculating loop, streaking on prepared NA culture medium plate, and dark culturing at 25 deg.C; after the colonies grow out, selecting a faint yellow single colony, and performing Z-shaped plate drawing on another NA culture medium plate by using an inoculating loop. Three successive inoculations on NA plates, confirmed as a single colony, with no other bacteria co-growing, were considered to have obtained pure culture, designated ZN-S4.

The strain ZN-S4 is a Burkholderia gladioli strain, and the preservation information is as follows:

the preservation name is Burkholderia gladiolis, the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, kcgmcc NO:18963, time of deposit 2019, 11/18.

Example 2 identification of the strain ZN-S4:

the Burkholderia gladiolis ZN-S4 has the following characteristics: the suitable growth temperature of the strain on the NA culture medium is 23 ℃; the bacterial colony is light yellow, round, wet and sticky, smooth in boundary, short in rod shape, and negative in gram stain. The form is shown in figure 1. ZN-S4 strain obtains a gene sequence with 1386bp in full length through 16S rRNA sequencing, and homology comparison is carried out on BLAST to find that the similarity of the gene sequence and relevant sequences of Burkholderia gladioli (Burkholderia gladioli), burkholderia cepacia (Burkholderia cepacia), burkholderia farinosa (Burkholderia pseudomelina) and the like is over 99 percent. The phylogenetic tree is constructed by calculation by a Neihbor-Joining method, and the result shows that the genetic distance between the endophytic bacterium ZN-S4 and Burkholderia gladioli CIP105410 is closest, the endophytic bacterium ZN-S4 is gathered in one branch (figure 2), and the ZN-S4 is finally identified as Burkholderia gladioli (Burkholderia gladioli) by combining morphology.

Example 3 growth promoting effect of burkholderia gladioli ZN-S4 with different concentrations on rice seedlings:

selecting ZN-S4 single colony, inoculating the single colony to 100mL NB culture solution, placing the culture solution in a 23 ℃ environment, carrying out shaking culture at 180r/min, adjusting the OD value of bacterial solution to 1.0 at 600nm by using a spectrophotometer after 48h, diluting the bacterial solution to 50 times, 100 times, 200 times and 400 times by using sterile water, soaking rice seeds in a 30 ℃ artificial climate box, carrying out seed soaking and germination acceleration, carrying out seed soaking treatment for 48h, replacing the bacterial solution once every 24h, sowing the germinated rice seeds in pots with the length of 20cm multiplied by 15cm, sowing 3 rows and 10 plants in each row, arranging 1 variety (line) in each row, repeating and randomly arranging each treatment for 3 times, using the sterile water seed soaking treatment as a control, covering a film after the sowing is finished, carrying out moisture preservation, punching on the film, ventilating, placing the film in the artificial climate box, carrying out 28 ℃ culture for 3d, transferring the pots to a glass greenhouse for culture after emergence, watering once a day, carrying out normal management, and carrying out investigation on the growth promotion effect at the 30d after the sowing.

Measurement indexes are as follows:

(1) Seedling height and root length: 10 rice seedlings are taken from each variety of each pot, the plant height and the root length of each variety are measured by taking 30 rice seedlings from each variety, wherein the longest root is taken as a measuring object, and the average value of each variety of 30 seedlings treated is taken as the plant height and the root length (cm).

(2) Seedling dry weight and root dry weight: the seedlings and roots are separately packaged in small glass bottles, the bottles are put into a 60 ℃ oven to be dried to constant weight, then an analytical balance is used for weighing, and the dry weight (g) is obtained by dividing the weight by the number of seedlings.

The results show (see FIGS. 3-6): 200 times of the dilution (0.5X 10) ⁷ cfu/mL) has obvious growth promoting effect on 3 varieties (Yongyou 1540, zhongzhe you No.1 and Tailiangyou 217) of rice seedlings.

Example 4 Effect of different concentrations of Burkholderia gladioli ZN-S4 on tomato shoot Biomass

(1) The root irrigation treatment has the growth promoting effect on tomatoes:

preparation of potted tomato seedlings: floating the tested tomato seeds with clear water to remove shrunken grains, washing the seeds with running water for 2-3min, soaking the seeds with warm water for 24h, putting the seeds on sterile and moist gauze, placing the gauze in an artificial climate box at 30 ℃ for germination acceleration in a dark period for 2d, sowing the germinated seeds in a hole tray containing a sterile nutrient medium, watering once every 24h, and carrying out root irrigation treatment test after the 2 nd true leaf of the tomato grows out.

Preparing bacterial liquid and irrigating roots: and (3) selecting a single colony of ZN-S4, inoculating the single colony into 100mL of NB culture solution, placing the single colony into 23 ℃, performing shaking culture at 180r/min, adjusting the OD value of bacterial liquid to 1.0 by using a spectrophotometer at 600nm after 48h, and then diluting the bacterial liquid into 50-fold, 100-fold and 200-fold diluted liquid by using sterile water for later use. Transplanting tomato seedlings in the 2-leaf stage into a small pot from a plug tray, namely 1 pot for 1 seedling, irrigating roots with the prepared ZN-S4 diluted bacterial solution, irrigating 30mL of each pot, treating 18 seedlings, taking irrigated sterile water as a blank control, placing the treated seedlings in a room for 2d seedling revival, culturing the treated seedlings in a glass greenhouse, managing according to a conventional method, and investigating the growth promoting effect at the 21 st day after transplanting and root irrigating treatment.

The measuring index and method are as follows:

a. fresh weight of seedling and fresh weight of root: seedlings and roots were separated and weighed separately on a 0.001g scale, and the average value of each plant was taken as the fresh weight (g) of seedlings and the fresh weight (g) of roots.

b. Leaf dry weight of 3,4 true leaves: 2 true leaves (including petioles) and stems are separately packaged by a vessel, dried in a dryer at 60 ℃ to constant weight and weighed by a 0.001g balance, and the weight is divided by the number of seedlings to obtain dry weight (g).

c. Total number of blades: the number of fully developed leaves per tomato plant (without cotyledons) was recorded.

(2) Effect of spray treatment on growth and development of tomatoes:

culturing potted tomato seedlings: the method is the same as above, and the tomato seedlings are used for tests when growing to 3-leaf stage.

Preparing bacterial liquid and carrying out spray treatment: selecting activated ZN-S4 single colony, inoculating into 250mL conical flask containing 50mL NB culture medium, shake culturing, transferring the bacterial liquid into 50mL sterile centrifuge tube after 48 hr, centrifuging at 6000r/min for 10min, removing supernatant, suspending the bacterial with sterile 0.05% Tween-20, adjusting OD450nm value of bacterial suspension to 1.0 (10) ⁹ cfu/mL), and further diluting the bacterial suspension in 50-fold, 100-fold and 200-fold dilutions. Selecting tomato potted seedlings with consistent growth vigor and 3-leaf period, spraying once again at an interval of 24h for 2 times, treating 18 plants each time, and setting a blank control of 0.05% Tween-20; the growth promoting effect was investigated at 21d after the treatment, which was conducted according to the conventional method. The growth promoting effect was investigated at 21d after transplantation for 18 strains per treatment.

The measuring index and method are as follows: root irrigation experiment.

(3) Results of the experiment

ZN-S4 root irrigation treatment groups with various concentrations can obviously promote the growth of tomato seedling biomass (FIGS. 7 and 8). Wherein the growth promoting effect of 100 times of diluent treatment is most obvious and is obviously higher than that of a control group, and the fresh weight of seedlings, the fresh weight of roots, the dry weight of leaves and the total leaf number of the control group are respectively increased by 62.57%,88.68%,80.68% and 25.00% compared with the control group. In addition, the growth indexes of 50-fold, 100-fold and 200-fold dry weight of the removed leaves among 3 treatment groups have no significant difference, which indicates that the growth promoting capability of the ZN-S4 strain on tomato seedlings is not in a concentration effect relationship with bacterial liquid.

ZN-S4 leaf surface spray treatment groups with various concentrations can obviously promote tomato seedling growth (figure 9), wherein 50 times of diluent has the optimal growth promotion effect, various growth indexes are obviously higher than those of a control group, and the fresh weight, the fresh root weight, the dry leaf weight and the total leaf number of a single tomato seedling are respectively increased by 23.79%, 37.80%, 9.87% and 14.49% compared with a blank control. In addition, except for the fresh weight of a single root, growth indexes of 50-fold, 100-fold and 200-fold treatment groups have no significant difference, and the fact that the ZN-S4 strain has no concentration effect relationship on the growth promoting capacity of tomato seedlings and bacterial liquid is shown again.

Example 5 Effect of different concentrations of Burkholderia gladioli ZN-S4 on cucumber shoot Biomass

(1) Preparation of potted cucumber seedlings: floating the tested cucumber seeds with clear water to remove shrunken grains, flushing with running water for 1-2min, soaking the seeds with warm water for 24h, placing the soaked seeds on moist sterile gauze, placing the gauze in a 30 ℃ artificial climate box for germination for 1d in a dark period, sowing the germinated seeds in a hole tray containing a sterile nutrient medium, watering once every 24h, and carrying out root irrigation treatment tests when the cucumber grows to 2 leaves.

(2) The influence of the preparation of the bacterial liquid and the root irrigation treatment on the growth and development of the cucumber:

(1) and (2) selecting a single colony with normal ZN-S4 morphology, inoculating the single colony into 100mL of NB culture solution, placing the single colony in 23 ℃, carrying out shaking culture at 180r/min, adjusting the OD value of the bacterial liquid to 1.0 by using a spectrophotometer at 600nm after 48h, and diluting the bacterial liquid into 50-fold, 100-fold and 200-fold diluent by using sterile water for later use. Transplanting cucumber seedlings at the 2-leaf stage into a small pot from a plug tray, transplanting 1 seedling in 1 pot, irrigating roots with the prepared ZN-S4 diluted bacterial liquid, irrigating 30mL each pot, treating 18 seedlings each time, taking irrigated sterile water as blank control, placing the seedlings indoors for 2 days for slow seedling culture, culturing in a glass greenhouse, managing according to a conventional method, and investigating the growth promoting effect at 21d after transplanting and root irrigation treatment.

(2) The measuring index and method are as follows: the same tomato experiment is carried out.

(3) Effect of spray treatment on cucumber growth and development:

(1) cultivation of potted cucumber seedlings: the method is the same as the above, after the true leaves of the cucumber seedlings are unfolded, transplanting the cucumber seedlings into a small pot, 1 pot of the cucumber seedlings is transplanted into a glass greenhouse for conventional culture after the cucumber seedlings are placed indoors for seedling delay, and the cucumber seedlings are used for testing when the cucumber seedlings grow to the 3-leaf stage.

(2) Preparing bacterial liquid and carrying out spray treatment: the bacterial liquid preparation is the same as above. Selecting cucumber potted seedlings with consistent growth vigor and 3-leaf stage, spraying once again at intervals of 24h for 2 times, treating 18 plants each time, and setting 0.05% Tween-20 blank control; the growth promoting effect was investigated at 21d after the treatment, which was conducted according to the conventional method.

The measuring index and method are as follows: the same tomato experiment is carried out.

(4) Results of the experiment

(1) ZN-S4 root irrigation treatment groups with various concentrations can obviously promote the growth of the biomass of cucumber seedlings (FIGS. 10 and 11). Compared with a control, the 200-time diluent remarkably (P is less than 0.05) promotes the increase of the fresh weight of cucumber seedlings and the fresh weight of roots, and the increase rates are 11.62 percent and 20.00 percent respectively.

(2) ZN-S4 strain leaf surface spraying can obviously promote cucumber seedling growth (figure 12), 100 times and 200 times of treatment groups except total leaf number, all other biomass indexes are obviously higher than those of other treatment groups in the same period, wherein 100 times of diluent has the best growth promotion effect on seedling fresh weight, root fresh weight and leaf dry weight, the growth rate is large, and the growth promotion effect is increased by 23.68%,93.28% and 38.39% respectively compared with a control group.

Example 6 colonization of Burkholderia gladioli ZN-S4 in roots, stems and leaves of Rice

(1) Test method

(1) Induction of rifampicin-resistant marker strains

Rifampicin (Rif) antibiotic was added to NB medium to prepare a culture solution with a Rif concentration of 0.5. Mu.g/mL. Transferring the test strain ZN-S4 into the culture medium, placing in a proper temperature, and culturing at 180r/min for 36-48h to obtain a bacterial liquid for later use. Dipping the bacterial liquid by using an inoculating loop, transferring the mutant to an NA flat plate containing Rif with the same concentration by adopting a scribing method, subculturing for 1 time, selecting a single colony, transferring the single colony to a next Rif NB culture medium with high concentration of 1 mu g/mL, culturing, gradually increasing the Rif concentration according to a similar method, sequentially setting concentration gradients to be 0.5, 1, 2, 4, 8, 36, 64, 128 and 300 mu g/mL until a mutant strain ZN-S4t which can stably grow, has normal shape and unchanged antibacterial activity on the NA flat plate containing 300 mu g/mL Rif is screened, and carrying out permanent planting dynamic detection by taking the mutant strain as a marked strain.

(2) Colonization of marked strain on potted rice seedling

Preparation of potted rice seedlings: putting the Yongyou 1540 rice seeds in an artificial climate box at the temperature of 30 ℃ for sterile water soaking and germination acceleration for 3d; simultaneously sterilizing the cultivation soil by high-pressure steam (121 ℃, 110 KPa) for 50min and 24h, sterilizing again, cooling the soil, then potting and watering, sowing the germinated rice seeds in pot bowls with the length and width of 20cm multiplied by 15cm, sowing 20 seeds in each pot for 27 pots in total, covering the film and preserving moisture after the sowing is finished, puncturing holes on the film and ventilating, putting the pot into an artificial climate box for cultivation at 28 ℃ for 3 days, transferring the pots into a glass greenhouse for cultivation after the emergence of seedlings, watering once every other day, carrying out normal management, and supplying the rice seedlings for test when the rice seedlings grow to the 4-leaf stage.

Preparing bacterial liquid: the ZN-S4t strain is inoculated into a 500mL conical flask containing 150mL NB culture medium containing Rif, the temperature is suitable for culture, after 36h, the bacterial liquid is transferred to a 50mL sterile centrifuge tube and centrifuged at 6000r/min for 10min, the supernatant is discarded, the bacterial is suspended by sterile water and adjusted to bacterial suspension with OD450nm of 1.0 (109 cfu/mL) for standby.

Plant treatment: and (3) taking seedlings growing to 4-leaf stage and having consistent size, washing clean root systems with sterile distilled water, respectively placing the root systems in the prepared ZN-S4 mutant strain suspension liquid for root soaking treatment for 30min, then transplanting the seedlings into pots filled with 0.5kg of sterile cultivation soil, and sampling 1, 3, 5, 9, 15, 20 and 25d after transplanting to detect the quantity of ZN-S4t in each pot.

Detecting the quantity of ZN-S4 t: cutting rice root (1 cm below stem base), stem (1 cm above stem base), and leaf (1 cm above leaf tongue) 0.5g each, and washing with sterile waterWashing for 2-3 times. Soaking in 75% ethanol for 3min, washing with sterile water for 2 times, soaking in 0.1% sodium hypochlorite for 40s, washing with sterile water for 4 times, coating the NA plate with 100 μ L of the final sterile water washing solution, and culturing at 25 deg.C for 24h to ensure that no bacterial colony appears on the plate, which indicates that the tissue surface is thoroughly sterilized. Cutting rice leaves into small segments with sterile scissors, placing into a mortar, adding 5mL of sterile water and a little of sterile quartz sand, grinding into slurry, diluting with sterile water to 10% ^-1 、10 ^-2 、10 ^-3 Standing for 3-5min with three dilutions of different concentrations. mu.L of each dilution was pipetted onto NA plates (9 cm) containing 300. Mu.g/mL Rif and repeated 3 times for each treatment. Respectively placing in a proper temperature, culturing in dark for 36-48h, checking the colony number, and calculating the quantity of ZN-S4t in root, stem and leaf tissues per gram. And (3) determining the planting condition of the endophytic bacterium ZN-S4 in roots, stems and leaves of the rice.

Bacterial content per gram of tissue = colony number × dilution factor × water amount for separation/(plate water amount × tissue weight for separation) (2) experimental results:

FIGS. 13 and 14 show that ZN-S4t has strong colonization ability in rice plants and can be effectively colonized in rice roots and stems; the planting time is long, the planting quantity is stable, and the root planting bacterial quantity is still maintained at 2.77 multiplied by 10 when the test is stopped for 25 days ⁵ cfu/g. This shows that the strain has good planting ability and competitive advantage, and mutual benefit symbiosis relationship may exist between the strain and the rice root system.

Example 7 prevention and treatment effects of Burkholderia gladioli ZN-S4 bacterium liquid spray on rice bacterial leaf blight at tillering stage

(1) Preparation of potted rice seedlings:

putting the Yongyou 1540 rice seeds in an artificial climate box at 30 ℃ for sterile water soaking and germination acceleration for 3d; simultaneously sterilizing the cultivation soil by high-pressure steam (121 ℃, 110 KPa) for 50min and 24h, sterilizing again, cooling the soil, then potting and watering, sowing the germinated rice seeds in pot bowls with the length and width of 20cm multiplied by 15cm, sowing 20 seeds in each pot for 27 pots in total, covering the film and preserving moisture after the sowing is finished, puncturing holes on the film and ventilating, putting the pot into an artificial climate box for cultivation at 28 ℃ for 3 days, transferring the pots into a glass greenhouse for cultivation after the emergence of seedlings, watering once every other day, carrying out normal management, and supplying the rice seedlings for test when the rice seedlings grow to the 4-leaf stage.

(2) Preparing a pathogenic bacterium liquid:

picking single bacterial colony of bacterial leaf blight probacteria, inoculating the bacterial colony into a 250ml conical flask filled with 50ml of NB culture solution, carrying out constant temperature oscillation culture at 28 ℃ at 180r/min, transferring the bacterial liquid into a 50ml sterile centrifuge tube after 24h, centrifuging at 6000r/min for 10min, discarding supernatant, suspending the bacterial body by using sterile 0.05% Tween-20, and adjusting the OD450nm value of bacterial suspension of bacterial leaf blight probacteria to 1.0 for later use.

(3) Inoculation of bacterial blight of rice and investigation of disease spots:

the leaf cutting method is adopted to inoculate the bacterial leaf blight of the rice, the method of the reference method reaches the people, and the rice leaves with good growth are selected as the leaves to be inoculated. Dipping prepared bacterial liquid of bacterial blight of rice by sterilized surgical scissors, cutting off the leaf apex at a position 1.0cm away from the leaf apex and perpendicular to the main vein, wrapping the wound with absorbent cotton full of the bacterial liquid of bacterial blight of rice, covering with a film, and moisturizing, wherein each plant of rice is inoculated with one leaf, and 30 plants are inoculated in each treatment. The indoor temperature is kept at about 30 ℃, the relative humidity is kept at about 90%, and when the area of the CK-attacked leaves is about 1/2, the attack area of each treated leaf is measured, and the prevention and treatment effect is calculated.

The control effect calculation method comprises the following steps:

control effect = (control lesion length-treatment lesion length)/control lesion length x 100%

(4) The experimental results are as follows:

ZN-S4 can effectively control the disease condition of bacterial leaf blight of rice in the seedling stage, the control effect of each treatment group is obvious, but the difference between the treatment groups is not obvious (Table 1), and the bacterium has a certain control effect on the bacterial leaf blight of rice, wherein the highest control effect is 100 times of diluent, and the average control effect reaches 58.19%. The research result shows that the endophytic bacterium ZN-S4 can effectively inhibit the occurrence and development of rice bacterial blight disease and has a good protection effect.

TABLE 1 ZN-S4 prevention and control effect on bacterial leaf blight of rice at seedling stage

Note: different lower case letters indicate the same-column data-significantly different (ANOVA, duncan, P < 0.05) values: mean ± standard deviation.

Example 8 measurement of Nitrogen fixation and phosphorus solubilization of Burkholderia gladioli ZN-S4

(1) Effect on the expression of the nifH gene: separately picking ZN-S4 and control strain (ZN-S2 CGMCC NO. 18962) single colony to be cultured in NB culture solution for 24h, centrifuging, discarding supernatant, and treating OD of bacterial solution with sterile distilled water _450nm Adjusting to 0.5, inoculating the strain into Ashby nitrogen-free culture solution according to 0.5 percent of inoculation amount, taking inoculated equal amount of sterile water as a control, placing the culture solution at 23 ℃, performing shaking culture at 150r/min for 7d, observing the growth condition of the strain, and if the culture solution becomes turbid, indicating that the endophytic bacterium has nitrogen fixation capacity, otherwise, no nitrogen fixation effect exists.

(2) ZN-S4 PCR amplification was performed using primers 968-F (5 'AACGCGAAGAACCTTAC-3') and 1401-R (5 'CGGTGTGT ACAAGACCC-3') specific to the azotobacter nifH to obtain a target product of 450bp. And detecting the PCR product by 1% agarose gel electrophoresis to verify whether the nitrogen-fixing gene exists in the thallus. PCR reaction systems and reaction procedures were referenced and optimized.

And (3) PCR reaction system:

PCR reaction procedure: preheating for 3min at 94 ℃; denaturation 30s at 94 deg.C, annealing at 56 deg.C for 1min, and extension at 72 deg.C for 1min, for 30cycles; extension was carried out at 72 ℃ for 5min and at 4 ℃ for forever.

The amplification result is shown in FIG. 15, ZN-S4 in the figure has an obvious band at 450bp, which indicates that the bacterium has the nitrogen fixation effect; no corresponding band appeared in the control strain ZN-S2, indicating no nitrogen fixation.

(3) Determination of soil available phosphorus (sodium bicarbonate method extraction-molybdenum antimony colorimetric method determination)

The seedling pot is adopted to divide soil (500 g/pot), 2 groups of soil culture samples are designed in total, wherein one group is 100 times of diluent of ZN-S4 (the diluent preparation method is implemented as the same wayExample 3): root irrigation was performed at 20 mL/pot, and another group was blank. Each group of 3 parallels, the sampling time is 21d and 45d respectively; fertilizer: 40g/m ² (i.e., 0.5 g/pot); the water content of the soil is kept to be about 60 percent of the field water capacity in the test process.

Weighing 5.00g of naturally air-dried soil sample passing through 1mm sieve in a 200mL triangular flask, adding 100mL of 0.5mol/L sodium bicarbonate solution, adding phosphorus-free activated carbon, oscillating on an oscillator for 30min, taking out, and immediately filtering with filter paper (discarding the initial 7-8mL of filtrate). Sucking 10mL of filtrate into a 50mL volumetric flask, adding 5mL of molybdenum antimony sulfate anti-mixed color developing agent, shaking up, adding water to a constant volume, and then fully shaking up. And (4) carrying out color comparison at the wavelength of 660nm of a spectrophotometer.

Calculating the available phosphorus content in the soil according to the following formula:

in the formula: sample solution concentration mg/L-the concentration of phosphorus in the sample solution obtained by standard curve checking;

constant volume- -volume before colorimetry (50 mL)

Division multiple of-100/10

m- - -weighing the soil sample (g)

And (3) measuring results: as shown in FIG. 16, after the ZN-S4 solution is subjected to root irrigation treatment, the results of sampling tests at 21d (T-21) and 45d (T-45) respectively show that the ZN-S4 treatment can obviously improve the content of available phosphorus in soil and is beneficial to plant absorption. The content of available phosphorus in the soil gradually decreases along with the growth of plants, and after 45d, the concentration of the available phosphorus in the soil of the bacteria liquid treatment group is still significantly higher than that of the control group regardless of the fact that the concentration of the control group or the treatment group is significantly lower than that of the control group after 21 d.

(4) Determination of Nitrogen fixation Capacity (refer to determination of Total Nitrogen in Water alkaline Potassium persulfate digestion ultraviolet Spectrophotometry (HJ 636-2012)

Selecting ZN-S4 single colony, inoculating the single colony into an Ashby nitrogen-free culture solution according to the method in the embodiment (1), placing the culture solution at 23 ℃, and performing shaking fermentation culture at 180r/min for 7d;

centrifuging the ZN-S4 fermentation liquor obtained by the fermentation for 7d at 10 000r/min for 15min, measuring 1mL of supernatant fluid, placing the supernatant fluid in a 25mL volumetric flask, diluting the supernatant fluid to 10mL by using non-ammonia water, adding 5mL of alkaline potassium persulfate solution (prepared as follows), plugging a bottle stopper, wrapping the bottle stopper by using kraft paper, digesting the bottle stopper at the temperature of 121-124 ℃ for 45min, cooling, adding 1mL of HCl solution at the mol/L, fixing the volume to a marked line by using the non-ammonia water, and measuring the volume to be measured. 10mL of non-aqueous ammonia was used as a blank in place of the sample solution. ZN-S4 Nitrogen fixation Capacity is shown in Table 2 below. As can be seen, in the liquid fermentation medium, the nitrogen concentration in the fermentation liquor after the bacteria fix the nitrogen in the air reaches 20.860 +/-1.914 mg/L.

Alkaline potassium persulfate solution:

40.0g of potassium persulfate (obtained by recrystallization) is weighed and dissolved in 600ml of water (can be placed in a water bath at 50 ℃ and heated until the potassium persulfate is completely dissolved); an additional 15.0g of sodium hydroxide was weighed out and dissolved in 300ml of water. After the sodium hydroxide solution is cooled to room temperature, the two solutions are mixed to a constant volume of 1000ml, and the mixture is stored in a polyethylene bottle and can be stored for one week.

Example 9 comparison of Nitrogen fixation and phosphorus solubilization Capacity of different strains

According to the method described in example 8 (2) and (3), the existing strains such as CCTCC NO: M2014672 (No. C1), GDMCC No. 61168 (No. C2), CGMCC No.18775 (No. C3), CGMCC No.6492 (No. C4), NJAU-B8 (No. C5) and the like were compared with the strain ZN-S4 of example 1, and the measurement results of nitrogen-fixing ability and phosphorus-solubilizing ability (culture 21 d) are shown in Table 2 below.

TABLE 2 comparison of the phosphorus-solubilizing and nitrogen-fixing capacities of different strains

From table 2, it can be seen that: the phosphorus dissolving capacity of five strains C1-C5 is obviously poorer than that of the strain ZN-S4 in the example 1, and the concentration of effective phosphorus in soil is not obviously different from that of a control group. In an Ashby N-free culture medium, the C2, C3, C4 and C5 strains can not normally grow in the culture medium, and both the C1 and ZN-S4 can normally grow, and detection of fermentation liquor shows that the nitrogen content of the C1 strain fermentation liquor is 2.600mg/L, while the nitrogen content of the ZN-S4 strain is up to 20.860mg/L and is obviously higher than that of the C1 strain.

Finally, it is also noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> Zhejiang university of agriculture and forestry

<120> biocontrol plant endophytic bacterium ZN-S4 and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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aacgcgaaga accttac 17

<210> 2

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cggtgtgtac aagaccc 17

Claims (3)

1. Burkholderia gladioli (B)Burkholderia gladioli) ZN-S4, which is characterized in that: the preservation number is CGMCC NO:18963.

2. the Burkholderia gladioli (B), (C) and (E) of claim 1Burkholderia gladioli) ZN-S4, characterized in that: has control effect on bacterial leaf blight of rice.

3. The Burkholderia gladioli (B), (C) according to claim 2Burkholderia gladioli) ZN-S4, characterized in that: has the effect of preventing and treating bacterial leaf blight of rice in tillering stage.

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