AU2021100685A4 - Degrading bacteria for allelopathic autotoxic substances of tobacco, wettable powder of complex microbial agent and uses - Google Patents

Abstract

OF THE DISCLOSURE The present invention belongs to the technical field of the control of tobacco bacterial wilt, and in particular, relates to degrading bacteria for allelopathic autotoxic substances of tobacco, wettable powder of a complex microbial agent, and uses. According to the present invention, degrading bacteria capable of degrading the allelopathic autotoxic substances are screened, and finally six strains of the degrading bacterium are obtained specific to the allelopathic autotoxic substances including dibutyl phthalate, diisobutyl phthalate, benzoic acid, 3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde, or 2,4-ditertbutylphenol. By applying the degrading bacteria to the control of the tobacco bacterial wilt, substances harmful to the environment and produced from physical and chemical methods can be prevented; meanwhile, the degrading bacterium reduces the content of the allelopathic autotoxic substances in the soil by using allelopathic substances as nutrients, thereby reducing the growth stimulating effect of the allelopathic autotoxic substances on Ralstonia solanacearum and cutting off the chemotaxis inducing effect of the allelopathic autotoxic substances on the Ralstonia solanacearum. .. .. ... ....... [ 5FIG. ~FIG 22 241/2

Description

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DEGRADING BACTERIA FOR ALLELOPATHIC AUTOTOXIC SUBSTANCES OF TOBACCO, WETTABLE POWDER OF COMPLEX MICROBIAL AGENT AND USES BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention belongs to the technical field of the control of

tobacco bacterial wilt, and in particular, relates to degrading bacteria for allelopathic

autotoxic substances of tobacco, wettable powder of a complex microbial agent, and uses.

[0003] 2. Description of Related Art

[0004] Tobacco wilt has raised considerable attention in tobacco production, and

its control has become one of the research hotspots in fields related to tobacco production.

Current strategies for controlling bacterial wilt mainly include chemical control, biological

control, and agricultural control, wherein chemical control has the disadvantages of poor

late-stage preventive effect, and induction of chemical resistance of bacteria due to

long-term administration; and the low success rate of breed selection is the key problem in

agricultural control. Due to advantages such as safety, no pollution and long-acting effect,

biological control methods have become a hotspot in the control of tobacco bacterial wilt.

In research on tobacco bacterial wilt, it was found that the accumulation of allelopathic

autotoxic substances secreted by a root system is one of the causes of bacterial wilt.

Therefore, it is of great significance to prevent and treat bacterial wilt by reducing the

allelopathic autotoxic substances secreted by the root system and screening out a

degrading bacterium capable of degrading the allelopathic autotoxic substances.

BRIEF SUMMARY OF THE INVENTION

[0005] In view of the problems existing in the prior art, the present invention

provides a degrading bacterium for allelopathic autotoxic substances of tobacco, wettable powder of a complex microbial agent and uses, in order to solve or at least alleviate some of the problems in the prior art.

[0006] The present invention is implemented as follows: degrading bacteria for

allelopathic autotoxic substances of tobacco include at least one of a degrading bacterium

of Bacillus megaterium ED2 with an Accession No. of CCTCC M 2020589, or a

degrading bacterium of Bacillus amyloliquefaciens YZ1 with an Accession No. of CCTCC

M 2020593, or a degrading bacterium of Bacillus amyloliquefaciens B4 with an Accession

No. of CCTCC M 2020588, or a degrading bacterium of Bacillus sp. NO1 with an

Accession No. of CCTCC M 2020590, or a degrading bacterium of Bacillus sp. N013

with an Accession No. of CCTCC 2020591, or a degrading bacterium of Bacillus

amyloliquefaciens SD2 with an Accession No. of CCTCC M 2020592.

[0007] The degrading bacteria for the allelopathic autotoxic substances of the

tobacco involved in the present invention were preserved and survived in the China Center

for Type Culture Collection (zip code: 430072) of Wuhan University in Wuhan, China on

Oct. 16, 2010.

[0008] Further, the allelopathic autotoxic substances include at least one of dibutyl

phthalate, diisobutyl phthalate, benzoic acid, 3-methoxy-4-hydroxybenzoic acid,

p-hydroxybenzaldehyde, or2,4-ditertbutylphenol.

[0009] Further, the degrading bacterium of Bacillus megaterium ED2 degrades at

least one of the dibutyl phthalate or the p-hydroxybenzaldehyde, the degrading bacterium

of Bacillus amyloliquefaciens YZ1 degrades the diisobutyl phthalate, the degrading

bacterium of Bacillus amyloliquefaciens B4 degrades at least one of the benzoic acid or

the p-hydroxybenzaldehyde, the degrading bacterium of Bacillus sp. NO1 degrades the

3-methoxy-4-hydroxybenzoic acid, the degrading bacterium of Bacillus sp N013 degrades

the p-hydroxybenzaldehyde, and the degrading bacterium of Bacillus amyloliquefaciens

SD2 degrades the 2,4-di-tert-butylphenol.

[0010] A use of the degrading bacteria for the allelopathic autotoxic substances of

the tobacco as described in the degradation of the allelopathic autotoxic substances of the

tobacco is provided.

[0011] A use of the degrading bacteria for the allelopathic autotoxic substances of

the tobacco as described above in the preparation of a reagent for controlling tobacco

bacterial wilt is provided.

[0012] Wettable powder of a complex microbial agent includes the degrading

bacteria for the allelopathic autotoxic substances of the tobacco and powder additives.

[0013] Further, the powder additives include a carrier, a dispersant, a wetting agent,

a high-temperature stabilizer, and an ultraviolet protective agent.

[0014] Further, the carrier is white carbon black with an amount percentage of

%; the dispersant is sodium hexametaphosphate with an amount percentage of 2%; the

wetting agent is detergent powder with an amount percentage of 2%; the high-temperature

stabilizer is glycerin with an amount percentage of 1%; and the ultraviolet protective agent

is vitamin C with an amount percentage of 1%.

[0015] A use of the wettable powder of the complex microbial agent as described

above in the control of the bacterial wilt is provided.

[0016] Further, the bacterial wilt includes tobacco bacterial wilt.

[0017] A use of 2,4-ditertbutyl phenol, diisobutyl phthalate, dibutyl phthalate,

benzoic acid, p-hydroxybenzaldehyde, or vanillic acid in the preparation of a reagent for

promoting the growth of Ralstonia solanacearum is provided.

[0018] In summary, the present invention has the following advantages and

positive effects:

[0019] according to the present invention, degrading bacteria capable of degrading

the allelopathic autotoxic substances are screened, so that substances harmful to the

environment and produced from physical and chemical methods can be prevented; meanwhile, the degrading bacteria reduce the content of the allelopathic autotoxic substances in the soil by using allelopathic substances as nutrients, thereby reducing the growth stimulating effect of the allelopathic autotoxic substances on Ralstonia solanacearum and cutting off the chemotaxis-inducing effect of the allelopathic autotoxic substances on the Ralstonia solanacearum.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0020] FIG. 1 shows a liquid-phase degradation diagram of degrading bacteria for

allelopathic autotoxic substances;

[0021] FIG. 2 shows the antagonism of a dibutyl phthalate degrading bacterium

(left) and a benzoic acid degrading bacterium (right) against Ralstonia solanacearum;

[0022] FIG. 3 shows the antagonism of a diisobutyl phthalate degrading bacterium

against Ralstonia solanacearum; and

[0023] FIG. 4 shows a finished product of a complex microbial agent.

DETAILED DESCRIPTION OF THE INVENTION

[0024] To make the objects, technical solutions, and advantages of the present

invention clearer and more explicit, the present invention will be further illustrated below

in detail with reference to the embodiments. Devices and reagents used in the

embodiments and experimental examples are commercially available, unless otherwise

specified. The embodiments described herein are only intended to explain, instead of

limiting, the present invention.

[0025] Based on the information contained in this application, it is easy for those

skilled in the art to make various alternations to the precise description of the present

invention without departing from the spirit and scope of the appended claims. It should be

understood that the scope of the present invention is not limited to the defined processes, properties, or components, since these embodiments and other descriptions are merely intended to schematically illustrate specific aspects of the present invention. In fact, it is obvious that various alternations that can be made to the embodiments of the present invention by those skilled in the art or related arts should be covered within the scope of the appended claims.

[0026] To better understand the present invention instead of limiting the scope of

the present invention, all numbers representing dosages and percentages, and other

numerical values, as used in this application should be understood as being modified by

the word "about" in all cases. Therefore, unless otherwise specified, the numerical

parameters listed in the specification and the appended claims are approximate values,

which may be changed with different desired properties that are attempted to be obtained.

Each numerical parameter should be at least regarded as obtained based on the reported

significant figures and through conventional rounding methods. In the present invention,

"about" means within 10% and preferably 5% of a given value or range.

[0027] The present invention discloses degrading bacteria for allelopathic

autotoxic substances of tobacco, wettable powder of a complex microbial agent, and uses.

The details are as shown in the embodiments below. The degrading bacteria for the

allelopathic autotoxic substances of the tobacco involved in the present invention were

preserved in the China Center for Type Culture Collection of Wuhan University in Wuhan,

China, with the relevant information as follows: a degrading bacterium of Bacillus

megaterium ED2 with an Accession No. of CCTCC M 2020589, a degrading bacterium of

Bacillus amyloliquefaciens YZ1with an Accession No. of CCTCC M 2020593, a

degrading bacterium of Bacillus amyloliquefaciens B4 with an Accession No. of CCTCC

M 2020588, a degrading bacterium of Bacillus sp. NO1 with an Accession No. of CCTCC

M 2020590, a degrading bacterium of Bacillus sp. N013 with an Accession No. of

CCTCC 2020591, and a degrading bacterium of Bacillus amyloliquefaciens SD2 with an

Accession No. of CCTCC M 2020592.

[0028] Embodiment 1

[0029] 1. Components of allelopathic autotoxic substances

[0030] Tobacco field soil that had been continuously cropped for ten years was

used. Soil extracts were extracted with 80% ethanol solution. Acidic, neutral, and alkaline

components were separated and then detected by GC-MS, wherein the alkaline

components were mainly long-chain alkanes substances, which were not listed in Table 1.

The substances in Table 1 are those detected among the acidic and neutral components. A

relative response factor method was used as a quantitative method, and the calculation was

conducted based on a ratio of a peak area to a peak area of an internal standard compound.

Six substances were obtained, and the contents of the allelopathic substances increased

year by year due to continuous cropping.

[0031] Table 1 Content percentage (%) of allelopathic autotoxic substances

Content (%)

Non-co Continuous Continuo Continuo Continuo ntinuou Continuous cropping for 10 us us us Allelopathic substances s cropping year cropping cropping cropping croppin for 10 year (with serious for 1 year for 2 year for 4 year g diseases)

Benzoic acid - 0.01 0.05 0.14 0.16 0.20

3-methoxy-4-hydroxyben

zoic acid 0.03 0.11 0.12 0.17 0.22 0.18

P-hydroxy benzaldehyde 0.01 0.05 0.07 0.14 0.12 0.15

Diisobutyl phthalate 0.01 0.07 0.08 0.10 0.21 0.27

2,4-ditertbutylphenol - - 0.02 0.04 0.20 0.24

Dibutyl phthalate 0.08 0.23 0.3 0.37 0.52 0.67

[0032] 2. Screening of degrading bacteria for allelopathic autotoxic substances

[0033] Soil used for bacteria screening was collected from a sewage outlet in the

School of Life Sciences of Hubei University, silt from Shahu lake, and soil from a garbage

dump, and was transported back to the laboratory in triangular flasks. 10 g of the soil

collected from each site was placed into a sterilized triangular flask, added with 90 mL of

sterile water, and treated at 37°C and 230 rpm for 4 h. A dosed basic salt culture solution

containing allelopathic autotoxic substances was prepared by keeping each allelopathic

autotoxic substance in the culture solution at 100 mg/L; 10 ml of turbid soil solution was

added to 100 mL of the dosed culture solution, and cultured under the conditions of 37°C

and 230 rpm; after 5 days, 10 ml of bacterial suspension was taken and then added to 100

mL of the dosed culture solution again, and cultured for 5 days under the same conditions,

which was repeated for 3 cycles for acclimation. A bacterial solution obtained after the last

culture acclimation was spread on a basic salt culture dish containing the above

allelopathic substances, and incubated in a constant-temperature incubator at 37°C for 12

hours to observe the form and size of colonies. Colonies with distinct forms were selected

and scribed in an LB culture dish, and purification was conducted repeatedly to obtain a

single colony. The resulting bacteria were the degrading bacteria for the allelopathic

autotoxic substances.

[0034] 3. Detection of degradation efficiency of degrading bacteria

[0035] All the primarily screened degrading bacteria for the allelopathic autotoxic

substances were activated in an LB culture solution. 1 ml of a resulting bacterial solution

was inoculated in the dosed basic salt culture solution (50 ml), and cultured at 37°C and

230rpm for 48 hours. Then, allelopathic substances remaining in the culture solution were

extracted with an extraction method, and an extracted solution was filtered with a 0.22 m

filter membrane and added to a liquid-phase bottle. Afterwards, the degradation efficiency of the degrading bacteria was detected by high performance liquid chromatography. By comparing the sizes of the peak areas of the degrading bacteria, the best degrading bacterium with respect to all the allelopathic autotoxic substances was finally obtained.

[0036] Liquid-phase conditions:

[0037] Dibutyl phthalate and diisobutyl phthalate: mobile phases: methanol and

water at a ratio of 95:5; flow rate:1 mL/min; ultraviolet wavelength: 245 mm; injection

volume: 20 l; and column temperature: 300 C.

[0038] P-hydroxybenzaldehyde: mobile phases: methanol and water at a ratio of

:50; flow rate: 1 mL/min; ultraviolet wavelength: 227 mm; injection volume: 20 l; and

column temperature: 300 C.

[0039] 3-methoxy-4-hydroxybenzoic acid: mobile phases: methanol and 1%

formic acid aqueous solution at a ratio of 40:60; flow rate: 1 mL/min; ultraviolet

wavelength: 290 mm; injection volume: 10 l; and column temperature: 300 C.

[0040] Benzoic acid: mobile phases: methanol and 0.02 mol/L ammonium acetate

at a ratio of 5:95; flow rate:1 mL/min; ultraviolet wavelength: 230 mm; injection volume:

l; and column temperature: 250 C.

[0041] 2,4-ditertbutylphenol: mobile phase: methanol; flow rate: 1 mL/min;

ultraviolet wavelength: 275 mm; injection volume: 20 l; and column temperature: 300 C.

[0042] The degradation rate of the degrading bacteria was calculated as follows:

degradation rate = (initial concentration of allelopathic substances in the culture medium

concentration of remaining allelopathic substances in the culture medium) + initial

concentration of allelopathic substances in the culture medium x 100%

[0043] Experimental results: the peaks in a liquid phase diagram of degrading

bacteria with respect to various substances are shown in FIG. 1. In FIG. 1, a. represents a

residual amount of p-hydroxybenzaldehyde after degradation of a 3-hydroxybenzaldehyde

degrading bacterium; b. represents a residual amount of 3-methoxy-4-hydroxybenzoic acid after degradation of a 3-methoxy-4-hydroxybenzoic acid degrading bacterium; c.

represents a residual amount of 2,4-ditertbutylphenol after degradation by a

2,4-ditertbutylphenol degrading bacterium; d. represents a residual amount of diisobutyl

phthalate after degradation by a diisobutyl phthalate bacterium; e. represents a residual

amount of benzoic acid after degradation by a benzoic acid degrading bacterium; and f.

represents a residual amount of dibutyl phthalate after degradation by a dibutyl phthalate

degrading bacterium.

[0044] The 16s rRNAs of the degrading bacteria for the allelopathic and autotoxin

substances were compared through BLAST and identified by a development tree to obtain

genuses, as shown in Table 2 below.

[0045] Table 2 Degrading bacteria and degradation efficiency

Degradation Allelopathic substances Degrading bacteria efficiency

Dibutyl phthalate Bacillus megaterium ED2 89.3%

Diisobutyl phthalate Bacillus amyloliquefaciens YZ1 100%

Benzoic acid Bacillus amyloliquefaciens B4 82.35%

3-methoxy-4-hydroxybenzoic Bacillus sp. NO1 50.013% acid

P-hydroxy benzaldehyde Bacillus sp NO13 48%

2,4-ditertbutylphenol Bacillus amyloliquefaciens SD2 78.16%

[0046] In addition, to further detect whether the degrading bacteria were capable

of degrading a plurality of allelopathic substances at the same time, the obtained

degrading bacteria in Table 2 were inoculated to 1 mL of 100 mg/L dosed basic salt

culture solution (50 mL), respectively, and cultured at 37°C and 230 rpm for 48 hours.

Then, allelopathic substances remained in the culture solution were extracted with an

extraction method, and an extracted solution was filtered with a 0.22 m filter membrane and added to a liquid-phase bottle. Afterwards, the degradation efficiency of the degrading bacteria for other allelopathic substances was detected by high performance liquid chromatography.

[0047] Experimental results showed that the benzoic acid degrading bacterium B4

can simultaneously degrade p-hydroxybenzaldehyde, with the degradation efficiency of

11.13±0.25%; the dibutyl phthalate degrading bacterium of Bacillus megaterium ED2 can

also simultaneously degrade p-hydroxybenzaldehyde, with the degradation efficiency of

33.50±1.08%.

Embodiment 2

[0048] 1. Antagonistic effect of degrading bacteria on tobacco Ralstonia

solanacearum

[0049] Ralstonia solanacearum HF1-1 was provided by the Microbiology

Laboratory of the School of Life Science of Hubei University.

[0050] The degrading bacteria for various allelopathic autotoxic substances were

activated with an LB medium, and the Ralstonia solanacearum was activated with an NA

culture solution. An NA plate containing the spread Ralstonia solanacearum was

perforated, and a solution of each degrading bacterium was added to each well to detect an

antagonism between the degrading bacterium and the Ralstonia solanacearum through a

flat plate confrontation experiment.

[0051] FIG. 2 shows the antagonism of a dibutyl phthalate degrading bacterium

(left) and a benzoic acid degrading bacterium (right) against the Ralstonia solanacearum.

FIG. 3 shows the antagonism of a diisobutyl phthalate degrading bacterium against the

Ralstonia solanacearum.

[0052] Experimental results show that the dibutyl phthalate degrading bacterium,

the diisobutyl phthalate degrading bacterium, and the benzoic acid degrading bacterium

were obviously antagonistic to the Ralstonia solanacearum, and no such property was found in other bacteria.

[0053] 2. Control effect of degrading bacteria on tobacco bacterial wilt

[0054] Six types of degrading bacteria were prepared into bacterial suspensions

and then irrigated to potted tobacco seedlings. 3 treatment groups were set as follows: TI:

a group added with Ralstonia solanacearum; T2: a group added with Ralstonia

solanacearum and allelopathic and autotoxin substances; T3: a group added with Ralstonia

solanacearum, allelopathic and autotoxin substances, and degrading bacterium. The

concentration of a Ralstonia solanacearum solution irrigated to each group was 5x107

CFU/mL, the concentration of the allelopathic and autotoxin substances was 100 mg/L,

and the concentration of the degrading bacterium was 5x107 CFU/mL. The tobacco

seedlings were inoculated with the Ralstonia solanacearum and the allelopathic and

autotoxin substances for 48 hours, and then inoculated with the degrading bacteria for the

allelopathic and autotoxin substances. The symptoms of tobacco wilt were observed on

Day 7 after inoculation. The groups were treated in triplicate as a parallel, with 12 tobacco

seedlings treated in each group.

[0055] The specific data of the experimental results are shown in Table 3 below.

When the allelopathic and autotoxin substances were added, the incidence rate of the

bacterial wilt could be increased, and after the degrading bacteria for the allelopathic and

autotoxin substances were added, the incidence rate of the bacterial wilt was reduced.

After the treatment with Bacillus megaterium ED2, the incidence rate was reduced by

43.29% compared with that of the dibutyl phthalate treatment group of T2; after the

treatment with Bacillus amyloliquefaciens YZ1, the incidence rate was reduced by 50.78%

compared with that of the diisobutyl phthalate treatment group of T2; after the treatment

with Bacillus amyloliquefaciens B4, the incidence rate was reduced by 48.41% compared

with that of the benzoic acid treatment group of T2; after the treatment with Bacillus sp.

NO1, the incidence rate was reduced by 25.09% compared with that of the

3-methoxy-4-hydroxybenzoic acid treatment group of T2; after the treatment with Bacillus

sp. NO13, the incidence rate was reduced by 29.24% compared with that of the

p-hydroxybenzaldehyde treatment group of T2; and after the treatment with Bacillus

amyloliquefaciens SD2, the incidence rate was reduced by 26.45% compared with that of

2,4-ditertbutylphenol treatment group of T2.

[0056] It indicates that the degrading bacteria for the allelopathic and autotoxin

substances can effectively alleviate the accumulation of the allelopathic and autotoxin

substances, reduce the occurrence of the bacterial wilt, and have a certain effect on the

control of the bacterial wilt in the field.

[0057] Table 3 Control of bacterial wilt by the allelopathic and autotoxin

substances and the degrading bacteria

Treatment group Incidence rate

TI 48.68% 6.5%

T2-Dibutyl phthalate 69.39% 10.9%

T3-Bacillus megaterium ED2 39.35% 9.2%

T2-Diisobutyl phthalate 57.30% 5.9%

T3-Bacillus amyloliquefaciens YZ1 28.20% 4.62%

T2-Benzoic acid 68.91%± 11.24%

T3-Bacillus amyloliquefaciens B4 35.55%± 8.69%

T2-3-methoxy-4-hydroxybenzoic acid 54.33%± 7.2%

T3-Bacillus sp. NO1 40.7%± 6.32%

T2-P-hydroxy benzaldehyde 90.3%±3.47%

T3-Bacillus sp NO13 63.9%±10.22%

T2-2,4-ditertbutylphenol 50.78%±3.91%

T3-Bacillus amyloliquefaciens SD2 37.35% ±6.88%

[0058] 3. Induction experiments of growth stimulation and chemotaxis induction of Ralstonia solanacearum by allelopathic autotoxic substances

[0059] A sterile capillary tube with an inner diameter of 1 mm was used, pipetted

with each of 6 types of allelopathic substance solutions with a concentration of 100

[imol/L, vertically immersed in a culture dish of a Ralstonia solanacearum solution, and

stood for 40 minutes. Liquid in the capillary tube was removed with a syringe, diluted to

1x10- 5 , spread on an NA solid plate containing TTC, and cultured at 30°C for 18 h, and

then, counting was conducted. Each treatment was repeated 3 times, with a PBS buffer as

a control. By treating with CK, dibutyl phthalate, diisobutyl phthalate, benzoic acid,

3-methoxy-4-hydroxybenzoic acid, p-hydroxybenzaldehyde and 2,4-ditertbutylphenol, the

colony numbers of the Ralstonia solanacearum were 3.63x106 cfu/mL, 2.03x10 7 cfu/mL,

1.0x107cfu/mL, 6.2x10 6cfu/mL, 3.63x10 6cfu/mL, 1.1x10 7 cfu/mL, 7.6x10 6 cfu/mL, and

1.93x10 7cfu/mL, respectively. This experiment demonstrated that the allelopathic

substances showed chemotactic effects on the Ralstonia solanacearum.

[0060] Effects of the allelopathic substances on the growth of Ralstonia

solanacearum: NA liquid media with different concentrations for the allelopathic autotoxic

substances were prepared, and then each inoculated with 1 mL of a Ralstonia

solanacearum solution (1.0x109 CFU/mL), and cultured at 30°C for 12 hours to obtain a

bacterial suspension. The bacterial suspension was diluted 10 times, and the concentration

(CFU/mL) of the Ralstonia solanacearum solution was measured at a wavelength of 600

nm. Experimental results are shown in Table 4 below: 2,4-ditertbutylphenol at 0.8 mg/L

shows the greatest effect on promoting the growth of the Ralstonia solanacearum;

diisobutyl phthalate at 1.5 mg/L shows the greatest effect on promoting the growth of the

Ralstonia solanacearum; dibutyl phthalate at 3.5mg/L shows the greatest effect on

promoting the growth of the Ralstonia solanacearum; benzoic acid at 0.3 mg/L shows the

greatest effect on promoting the growth of the Ralstonia solanacearum;

p-hydroxybenzaldehyde at 0.02mg/L shows the greatest effect on promoting the growth of the Ralstonia solanacearum; and vanillic acid at 0.1mg/L shows the greatest effect on promoting the growth of the Ralstonia solanacearum.

[0061] Table 4 Induction experiments of growth stimulation and chemotaxis

induction of Ralstonia solanacearum by allelopathic autotoxic substances

Effects of metabolites on the biomass of Ralstonia solanacearum Metabolites Metabolite concentration

2,4-ditertbutylphenol 0.0 mg/L 0.4 mg/L 0.6 mg/L 0.8 mg/L 1 mg/L 1.2 mg/L

OD600 1.43±0.07 1.56±0.021 2.54±0.09 3.62±0.05 1.19±0.03 1.01±0.04

Diisobutyl phthalate 0.Omg/L 0.5mg/L 1mg/L 1.5mg/L 2mg/L 2.5mg/L

OD600 1.43±0.07 3.623±0.02 3.93±0.02 4.17±0.04 4.02±0.02 4±0.02

Dibutyl phthalate 0.Omg/L 2mg/L 2.5mg/L 3mg/L 3.5mg/L 4mg/L

OD600 1.43±0.07 4.6±0.11 5.33±0.16 6.57±0.21 9.17±0.19 5.5±0.23

Benzoic acid 0.Omg/L 0.1mg/L 0.2mg/L 0.3mg/L 0.4mg/L 0.5mg/L

OD600 1.43±0.07 2.07±0.02 2.37±0.04 2.62±0.075 1.99±0.03 1.54±0.02

P-hydroxy 0.Omg/L 0.01mg/L 0.02mg/L 0.03mg/L 0.04mg/L 0.05mg/L benzaldehyde

OD600 1.43±0.07 3.92±0.01 4.62±0.02 4.11±0.021 3.95±0.03 3.83±0.01

Vanillic acid 0.Omg/L 0.1mg/L 0.2mg/L 0.3mg/L 0.4mg/L 0.5mg/L

OD600 1.43±0.07 1.85±0.11 1.81±0.06 1.64±0.07 1.54±0.02 1.26±0.02

[0062] Embodiment 3

[0063] 1. Preparation of wettable powder of complex microbial inoculant

[0064] On the basis of Embodiment 1 and Embodiment 2, in order to facilitate the

transportation and storage of the degrading bacteria, each of the degrading bacteria was

prepared into a complex microbial agent, the control effect of each complex microbial

agent on the tobacco bacterial wilt was observed.

[0065] Preparation of a complex microbial agent:

[0066] Each strain of Bacillus megaterium ED2, Bacillus amyloliquefaciens YZ,

Bacillus sp. NO1, Bacillus sp. N013, Bacillus amyloliquefaciens SD2, and Bacillus

amyloliquefaciens B4 was fermented with a soybean meal medium.

[0067] Various additives used in the wettable powder are as follows:

[0068] carrier: bentonite, diatomite, white carbon black, or talcum powder;

[0069] dispersant: sodium lignosulfonate, sodium pyrophosphate, sodium

hexametaphosphate, or PEG8000;

[0070] wetting agent: Shikakai Powder, nekal, or detergent powder;

[0071] high-temperature stabilizer: sodium carboxymethylcellulose, xanthan gum,

and glycerin; and

[0072] ultraviolet protective agent: citamin C and dextrin.

[0073] 70 mL of fermentation filtrate and 10 g of carrier were mixed and dried,

and then pulverized to obtain the master powder of a microbial agent. The master powder

and respective additives were mixed to detect a suspension rate, a suspension time, and a

spore content.

[0074] Table 5 Experimental results under conditions with different carriers

Spore content Spore content after 10 Carrier Wetting time (10 10cfu/g) days (10 1 °cfu/g)

Bentonite 44s 1.41 0.68

Diatomite 20s 2.54 1.71

Talcum powder 96s 0.42 0.12

White carbon black 14s 3.99 3.07

[0075] Table 6 Experimental results under conditions with different dispersants

Suspension Dispersant Wetting time percentage

Sodium 5 s 39.11% pyrophosphate

PEG8000 6s 40.22%

Sodium lignin 8s 39.77% sulfonate

Sodium

hexametaphospha 6s 68.80%

te

[0076] Table 7 Experimental results under conditions with different wetting agents

Suspension Wetting agent Wetting time percentage

Nekal 3 s 43.56%

Shikakai powder 4 s 40.22%

Detergent powder 4 s 60.22%

[0077] Table 8 Experimental results under conditions with different

high-temperaturestabilizers

Spore content after Spore content before High-temperature heat storage at 60°C heat storage stabilizer for 15 days (101 0 cfu/g)

(10 10 cfu/g)

Sodium

carboxymethylcellul 3.81 1.61

ose

Glycerol 4.01 3.12

Xanthan gum 3.87 0.36

[0078] Table 9 Experimental results under conditions with different ultraviolet

protective agents

Spore content after Spore content after

Ultraviolet protective ultraviolet radiation ultraviolet radiation

agent for 12 hours for 24 hours

( 10 °cfu/g) ( 1 0 1°cfu/g)

VC 2.99 2.49

Dextrin 2.01 0.99

[0079] A finished product of the prepared complex microbial agent is shown in

FIG. 4.

[0080] From the above experimental results, white carbon black was finally

selected as the carrier, accounting for 20%; sodium hexametaphosphate was selected as

the dispersant, accounting for 2%; detergent powder was selected as the wetting agent,

accounting for 2%; glycerin was selected as the high-temperature stabilizer, accounting for

1%; and VC was selected as the ultraviolet protective agent, accounting for 1%. The

specific steps are as follows.

[0081] After each degrading bacterium was fermented separately, all the

fermentation broths were controlled so that the colony after dilution and spreading was

about 4.09x109 CFU/mL; 10 mL of each of the fermentation broths was taken and

combined to reach 60 mL, mixed with 12 g of the carrier and then dried at 80°C for 16

hours to prepare the master powder of a microbial agent, which was diluted and spread to

detect the spore content therein, and the best carrier was selected. In case of the master

powder of the microbial agent with the white carbon black as the carrier, the best additives

were screened based on a mass ratio, with the dispersant and the wetting agent each accounting for 2% of the master powder, and the high-temperature stabilizer and the ultraviolet protective agent each accounting for 1%. After combining all the obtained additives withthe masterpowder, the spore contentwas detected to be 3.7xl 1 0 CFU/mL.

[0082] 2. Biological control effect of complex microbial agent

[0083] The complex microbial agent powder was applied to the field to control the

bacterial wilt, and 3 treatment groups were set as follows: T1: a group added with

Ralstonia solanacearum; T2: a group added with Ralstonia solanacearum and allelopathic

autotoxic substances; T3: a group added with Ralstonia solanacearum, allelopathic

autotoxic substances and complex microbial agent. The concentration of the Ralstonia

solanacearum solution for irrigating each group was 5x107 CFU/mL; the concentration of

the allelopathic autotoxic substances (a mixture of six substances) was 100 mg/L; and the

complex microbial agent powder was diluted 1000 times for irrigation so that the effective

bacterial solution therein is about 5x107 CFU/mL. Tobacco seedlings were inoculated with

the Ralstonia solanacearum and the allelopathic autotoxic substances, and after 48 hours,

inoculated with the complex microbial agent on Day 7 after inoculation, the symptoms of

tobacco wilt were observed, and the incidence rate of bacterial wilt was counted. The

groups were treated in triplicate as a parallel, with 12 tobacco seedlings treated in each

group.

Treatment group Incidence rate

T1 50.44% 7.55%

T2 80.90% 9.2%

T3 21.46% 4.42%

[0084] From the above results, it is known that the incidence rate in the case of the

complex microbial agent is 21.46% ±4.42%, which is 57.45% and 73.47% lower than that

of the groups T1 and T2, respectively, indicating that the complex microbial agent has a

stronger biological control effect than the administrated single degrading bacterium.

[0085] In summary, the degrading bacteria for the allelopathic and autotoxin

substances can effectively alleviate the high incidence rate of bacterial wilt caused by the

accumulated allelopathic autotoxic substances in the soil, and have a certain effect on

reducing the occurrence of the bacterial wilt. By preparing the degrading bacteria for the

allelopathic autotoxic substances into the complex microbial agent, the effect of the

degrading bacteria can be better exerted to achieve the effect of controlling the bacteria

wilt.

[0086] The description above is only preferred embodiments of the present

invention, but not intended to limit the present invention. Any modifications, equivalent

substitutions, improvements and the like made within the spirit and principle of the

present invention are construed to be incorporated within the protection scope of the

present invention.

Claims (10)

What is claimed is:

1. Degrading bacteria for allelopathic autotoxic substances of tobacco, characterized

by comprising at least one of a degrading bacterium of Bacillus megaterium ED2 with an

Accession No. of CCTCC M 2020589, or a degrading bacterium of Bacillus

amyloliquefaciens YZ1 with an Accession No. of CCTCC M 2020593, or a degrading

bacterium of Bacillus amyloliquefaciens B4 with an Accession No. of CCTCC M 2020588,

or a degrading bacterium of Bacillus sp. NO1 with an Accession No. of CCTCC M

2020590, or a degrading bacterium of Bacillus sp. N013 with an Accession No. of

CCTCC 2020591, or a degrading bacterium of Bacillus amyloliquefaciens SD2 with an

Accession No. of CCTCC M 2020592.

2. The degrading bacteria for the allelopathic autotoxic substances of the tobacco

according to Claim 1, characterized in that the allelopathic autotoxic substances comprise

at least one of dibutyl phthalate, diisobutyl phthalate, benzoic acid,

3-methoxy-4-hydroxybenzoicacid,p-hydroxybenzaldehyde,or2,4-ditertbutylphenol.

3. The degrading bacteria for the allelopathic autotoxic substances of the tobacco

according to Claim 2, characterized in that the degrading bacterium of Bacillus

megaterium ED2 degrades at least one of the dibutyl phthalate or the

p-hydroxybenzaldehyde, the degrading bacterium of Bacillus amyloliquefaciens YZ

degrades the diisobutyl phthalate, the degrading bacterium of Bacillus amyloliquefaciens

B4 degrades at least one of the benzoic acid or the p-hydroxybenzaldehyde, the degrading

bacterium of Bacillus sp. NO1 degrades the 3-methoxy-4-hydroxybenzoic acid, the

degrading bacterium of Bacillus sp. N013 degrades the p-hydroxybenzaldehyde, and the

degrading bacterium of Bacillus amyloliquefaciens SD2 degrades the

2,4-di-tert-butylphenol.

4. A use of the degrading bacteria for the allelopathic autotoxic substances of the

tobacco according to any one of Claims 1 to 3 in the preparation of a reagent for controlling tobacco bacterial wilt.

5. A use of 2,4-ditertbutyl phenol, diisobutyl phthalate, dibutyl phthalate, benzoic acid,

p-hydroxybenzaldehyde, or vanillic acid in the preparation of a reagent for promoting the

growth of Ralstonia solanacearum.

6. Wettable powder of a complex microbial agent, characterized by comprising the

degrading bacteria for the allelopathic autotoxic substances of the tobacco according

Claim 1 and powder additives.

7. The wettable powder of the complex microbial agent according to Claim 6,

characterized in that the powder additives comprise a carrier, a dispersant, a wetting agent,

a high-temperature stabilizer, and an ultraviolet protective agent.

8. The wettable powder of the complex microbial agent according to Claim 7,

characterized in that the carrier is white carbon black with an amount percentage of 20%;

the dispersant is sodium hexametaphosphate with an amount percentage of 2%; the

wetting agent is detergent powder with an amount percentage of 2%; the high-temperature

stabilizer is glycerin with an amount percentage of 1%; and the ultraviolet protective agent

is vitamin C with an amount percentage of 1%.

9. A use of the wettable powder of the complex microbial agent according to Claim 6

in the control of bacterial wilt.

10. The use according to Claim 9, characterized in that the bacterial wilt comprises

tobacco bacterial wilt.

FIG. 1

FIG. 2

FIG. 3 1/2