CN110804566B - Bacillus for preventing and treating root rot of paris polyphylla and preparation method and application thereof - Google Patents

Abstract

The invention discloses bacillus for preventing and treating paris polyphylla root rot and a preparation method and application thereof.A paenibacillus polymyxa strain HJ-2 is adopted to infect fusarium zoospores, so that the germination of fusarium spores is inhibited, and the harm of fusarium to rhizomes of paris polyphylla is reduced, so that the harm of root rot to paris polyphylla is reduced, the yield of paris polyphylla is ensured, and the economic loss of pesticide and agricultural is reduced; and the invention adopts Paenibacillus polymyxa strain HJ-2 for biological prevention, does not generate pesticide residue, and has the characteristics of green, recyclable, good prevention and control effect, pesticide residue pollution avoidance and the like.

Description

Bacillus for preventing and treating root rot of paris polyphylla and preparation method and application thereof

Technical Field

The invention relates to the technical field of biocontrol bacteria, in particular to bacillus for preventing and treating paris polyphylla root rot and a preparation method and application thereof.

Background

Paris polyphylla is a Liliaceae (Liliaceae) Paris (Paris) plant, mostly grows at the shady and warm place in the forest with the altitude of 800-1300 m, is distributed in the county, bamboo mountain, bamboo stream, Wudangshan and Fulongshan (Saiwang) in Shiweishan city, and is the most distinctive medicinal material 'four one' in northwest of the Ebei province; the drug material of the paris polyphylla collected in the 2015 edition of Chinese pharmacopoeia is the dried rhizome of Yunnan paris polyphylla or paris polyphylla of liliaceae, and paris polyphylla is an important formula drug of various famous and precious Chinese patent drugs such as Yunnan baiyao, Qudesheng snake drug, Gongxuening and the like; the elevation of the Shiweir area is generally higher, the forest coverage rate is high, the planting area of the Paris polyphylla in the Shiweir and the peripheral county cities is the largest, and the Paris polyphylla in the Heilongshan mountain in the Shiweir city is listed as a geographical sign product by the country; under-forest planting of traditional Chinese medicinal materials becomes an important growth point in the economy of the Shiwein city, and the cultivation area of the local paris polyphylla reaches more than 3000 mu;

root rot is one of the most important diseases of paris polyphylla, belongs to soil-borne diseases, is transmitted by taking soil as a medium, is particularly serious in humid and rainy seasons, is easy to cause large-area spread so as to cause the paris polyphylla dead production, seriously influences the yield, limits the development of the traditional Chinese medicine industry and causes great economic loss; currently, fusarium is found to be the main pathogenic bacterium causing root rot of paris polyphylla, and research shows that 5 fusarium (f.solani f.sp. paris, f.graminearum f.sp. paris, f.oxysporum, f.equiseti, f.tricinctum) can infect rhizome parts of paris polyphylla to cause root rot;

at present, the root rot prevention measure is to spray carbendazim or chemical agents to disinfect soil, but the effect is not ideal, the prevention and control for many years leads to the increase of the drug resistance of pathogenic bacteria, the soil degradation, the continuous cropping obstacle, the excessive carbendazim residue and other problems to be serious, and the yield and the quality of paris polyphylla are affected; therefore, the discovery of strains with biocontrol potential and the research and development of biological bactericides capable of replacing chemical bactericides have great significance for the control of the root rot of Paris polyphylla.

Disclosure of Invention

Aiming at the existing problems, the invention aims to provide the bacillus for preventing and treating the root rot of paris polyphylla and the preparation method and the application thereof, and the bacillus for preventing and treating the root rot of paris polyphylla is characterized in that the bacillus polymyxa strain HJ-2 is adopted to inhibit the germination of zoospores of fusarium (F.oxysporum), so that the harm of fusarium to rhizomes of paris polyphylla is reduced, the yield of paris polyphylla is ensured, and the economic loss of pesticide and agricultural is reduced; and the paenibacillus polymyxa strain HJ-2 is adopted for biological prevention, so that the paenibacillus polymyxa strain has the characteristics of being green and recyclable and avoiding pesticide residue pollution.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the bacillus strain is named as (Paenibacillus Polymyxa HJ-2), and is preserved in China center for type culture Collection in 8 and 9 months in 2019 with the preservation number of CCTCC NO. M2019617. And (4) storage address: south-east lake No. 8 of Wuchang district in Wuhan city, Hubei province.

Preferably, said HJ-2 is characterized by a growth phenotype: after culturing for 48h at 25 ℃ on a PDA culture medium, a colorless transparent HJ-2 colony is formed, and the colony is wet, sticky and neat in edge.

Preferably, the biochemical phenotype of HJ-2 is characterized by: decomposing glucose, sucrose, arabinose, xylose, mannitol, glycerol and lactose; sodium citrate and sodium succinate were negative with the test; does not generate H₂S; positive catalase test and negative oxidase test; positive in nitric acid reduction test, casein hydrolysis test, starch hydrolysis test and V-P test.

Preferably, the 16S rDNA sequence of HJ-2 is shown in seq ID No. 1.

Preferably, the preparation method of the paenibacillus polymyxa strain HJ-2 comprises the following steps:

the method comprises the following steps: isolation of Paenibacillus polymyxa Strain HJ-2

(1) Collecting a soil sample: collecting healthy paris polyphylla rhizosphere soil with the depth of 8-12cm from paris polyphylla planting bases in Shiwein city of Hubei, and bringing the healthy paris polyphylla rhizosphere soil back to a laboratory;

(2) putting 1g of rhizosphere soil into a 250ml sterilized triangular flask, adding 100ml of sterile water, sterilizing 30-40 glass beads, shaking on a shaking table at 160rpm for 30min, and standing for 30 min;

(3) taking supernatant, diluting to 10% by gradient dilution method^-4、10^-5、10-⁶Three gradients, respectively absorbing 0.1ml of liquid from the three gradients, inoculating the liquid to a beef extract peptone solid culture medium, and culturing at the constant temperature of 30 ℃ for 48 hours;

(4) selecting single colonies with different culture characteristics, selecting single colonies with different colony morphologies for streaking and purifying, transferring the purified strain to an NA slant culture medium, and storing at 4 ℃ for later use;

step two: identification of Paenibacillus polymyxa strain HJ-2

(1) Streaking colony morphology on a streaked and purified Paenibacillus polymyxa strain HJ-2 plate, wherein the HJ-2 colony cultured by a culture medium is circular, milky white, semitransparent, rod-shaped and spore-containing;

(2) further carrying out molecular identification according to the following steps:

a. firstly, extracting total DNA of a strain, adopting a 16SrDNA universal primer of bacteria, carrying out PCR amplification by taking genome DNA as a template, and carrying out agarose gel electrophoresis on an amplification product;

b. then, recovering and purifying a PCR product by using a glue recovery kit;

c. finally, cloning and transforming, screening positive clone sub-colonies, and sequencing after amplification culture;

preferably, the paenibacillus polymyxa is applied by the following specific steps:

the method comprises the following steps: activating the strain HJ-2, inoculating into 250mL triangular flask containing 50mL fermentation medium, shake culturing at 28 deg.C and 150rpm for 2 days to make the concentration of the bacterial liquid reach 2.0 × 10⁸cfu/ml—5.0×10⁸cfu/mL, centrifuging the fermentation liquid at 12000rpm for 5min, and filtering with a 0.22 μm bacterial filter to obtain sterile fermentation filtrate;

step two: the concentration obtained in the first step is 2.0 multiplied by 10⁸cfu/mL—5.0×10⁸The cfu/mL liquid microbial inoculum adopts a root irrigation method to prevent and control the root rot of the paris polyphylla at the early emergence stage of the paris polyphylla, and the root irrigation is carried out once in 7 days for 3 times continuously.

The invention has the beneficial effects that:

according to the bacillus for preventing and treating root rot of paris polyphylla, the preparation method and the application thereof, the paenibacillus polymyxa strain HJ-2 is adopted to inhibit the germination of fusarium (F. oxysporum) zoospores, and the harm of fusarium to rhizomes of paris polyphylla is reduced, so that the yield of paris polyphylla is ensured, and the economic loss of pesticide farmers is reduced; and the paenibacillus polymyxa strain HJ-2 is adopted for biological prevention, so that the paenibacillus polymyxa strain has the characteristics of being green and recyclable and avoiding pesticide residue pollution.

Drawings

FIG. 1-1 is a morphological diagram of a colony of Paenibacillus polymyxa HJ-2.

FIG. 1-2 is an electrophoretogram of HJ-2 strain 16 srDNA.

FIG. 2 is a phylogenetic tree diagram of Paenibacillus polymyxa HJ-2.

FIG. 3 is a schematic diagram of the experiment of the Paenibacillus polymyxa HJ-2 and Paris polyphylla five pathogenic bacteria confrontation, wherein: FIG. 3(a) is a schematic diagram of the experiment of the Paenibacillus polymyxa HJ-2 confronting pathogenic bacteria F.graminearum f.sp.paris; FIG. 3(b) is a schematic diagram of the experiment of Paenibacillus polymyxa HJ-2 and pathogenic bacteria F.solani f.sp.paris confrontation; FIG. 3(c) is a schematic diagram showing the experiment of the Paenibacillus polymyxa HJ-2 confrontation with pathogenic bacteria F.oxysporum; FIG. 3(d) is a schematic diagram of the experiment of the Paenibacillus polymyxa HJ-2 confrontation with pathogenic bacteria F. tricinctum; FIG. 3(e) is a schematic diagram showing the experiment of the Paenibacillus polymyxa HJ-2 and the pathogenic bacteria F.equiseti.

Fig. 4 is a schematic diagram showing the inhibition of f.oxysporum spore germination by paenibacillus polymyxa HJ-2, wherein: FIG. 4(a) is a schematic diagram showing germination of F.oxysporum spores without using Paenibacillus polymyxa HJ-2; FIG. 4(b) is a schematic view showing germination of F. oxysporum spores when Paenibacillus polymyxa HJ-2 was used.

FIG. 5 is a comparison chart of Paenibacillus mucilaginosus HJ-2 for controlling root rot of Paris polyphylla, wherein: FIG. 5(a) root irrigation experiment (Nutrition bowl, control); FIG. 5(b) root irrigation experiment (nutrition pot, HJ-2), FIG. 5(c) root irrigation experiment (plot, control); FIG. 5(d) root irrigation experiment (nutrition pot, HJ-2).

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following further describes the technical solution of the present invention with reference to the embodiments.

Referring to fig. 1-1, fig. 1-2, fig. 3(a), fig. 3(b), fig. 3(c), fig. 3(d), fig. 3(e), fig. 4(a), fig. 4(b), fig. 5(a), fig. 5(b), fig. 5(c) and fig. 5(d), a bacillus for controlling root rot of paris polyphylla, which is paenibacillus polymyxa HJ-2, is isolated from rhizosphere soil of paris polyphylla in decaweir, north lake, and has been preserved in the preservation Center of China Type Culture Collection (CCTCC) in marten university, marten, north lake, with the preservation number of CCTCC No. m 2019617, and the preservation address: south-east lake No. 8 of Wuchang district in Wuhan city, Hubei province.

The growth phenotype characteristics of the HJ-2 are as follows: culturing on PDA culture medium at 25 deg.C for 48 hr to form colorless transparent HJ-2 colony, which is wet and sticky and has uniform edge; the biochemical phenotypic characteristics of the HJ-2 are as follows: decomposing glucose, sucrose, arabinose, xylose, mannitol, glycerol and lactose; sodium citrate and sodium succinate were negative with the test; does not generate H₂S; positive catalase test and negative oxidase test; positive in nitric acid reduction test, casein hydrolysis test, starch hydrolysis test and V-P test;

the 16S rDNA sequence of the HJ-2 is shown as seq ID No. 1;

through 16SrDNA sequence comparison and analysis, the Paenibacillus polymyxa HJ-2 has high homology with Paenibacillus polymyxa (Paenibacillus polymyxa), the homology is 99%, and the Paenibacillus polymyxa belongs to the genus Bacillus (Paenibacillus) and is determined to be the Paenibacillus polymyxa (Paenibacillus polymyxa) by combining morphological characteristics, growth conditions and physiological and biochemical identification results of bacteria;

morphological characteristics of Paenibacillus polymyxa HJ-2: bacterial colonies of the strain are white, opaque, raised at the periphery and gram-positive.

The physiological and biochemical characteristics of the paenibacillus polymyxa HJ-2 are as follows: the results are shown in Table 1:

table 1: physiological and biochemical characteristics of bacillus HJ-2

Test items	Results	Test items	Results
				Salt tolerance		Sucrose	+
2％	+	Utilization of sodium citrate	－
				5％	－	Utilization of sodium succinate	－
10％	－	H2S production	－
				Sugar fermentation		Anaerobic growth	+
D-glucose	+	Contact enzyme	+
				L-arabinose	+	Oxidase enzyme	－
D-xylose	+	Reduction reaction of nitric acid	+
				Mannitol	+	Hydrolyzed casein	+
Glycerol	+	Hydrolyzed starch	+
				Lactose	+	V-P reaction	+

As can be seen from the experimental results in Table 1, the strain HJ-2 can reduce nitrate, hydrolyze starch, liquefy gelatin and the like, and the HJ-2 strain can be determined to belong to Paenibacillus polymyxa by looking up a table according to the identification results and combining gram staining observation results.

Example one, the preparation of the Paenibacillus polymyxa strain HJ-2 comprises the following steps:

the method comprises the following steps: isolation of Paenibacillus polymyxa Strain HJ-2

(1) Collecting a soil sample: collecting healthy paris polyphylla rhizosphere soil with the depth of 8-12cm from paris polyphylla planting bases in Shiwein city of Hubei, and bringing the healthy paris polyphylla rhizosphere soil back to a laboratory;

(2) putting 1g of rhizosphere soil into a 250ml sterilized triangular flask, adding 100ml of sterile water, sterilizing 30-40 glass beads, shaking on a shaking table at 160rpm for 30min, and standing for 30 min;

(3) taking supernatant, diluting to 10% by gradient dilution method^-4、10^-5、10-⁶Three gradients, respectively absorbing 0.1ml of liquid from the three gradients, inoculating the liquid to a beef extract peptone solid culture medium, and culturing at the constant temperature of 30 ℃ for 48 hours;

(4) selecting single colonies with different culture characteristics, selecting single colonies with different colony morphologies for streaking and purifying, transferring the purified strain to an NA slant culture medium, and storing at 4 ℃ for later use;

step two: identification of Paenibacillus polymyxa strain HJ-2

(1) Streaking colony morphology on a streaked and purified Paenibacillus polymyxa strain HJ-2 plate, wherein the HJ-2 colony cultured by a culture medium is circular, milky white, semitransparent, rod-shaped and spore-containing;

(2) further carrying out molecular identification according to the following steps:

a. firstly, extracting total DNA of a strain, adopting a 16SrDNA universal primer of bacteria, carrying out PCR amplification by taking genome DNA as a template, and carrying out agarose gel electrophoresis on an amplification product;

b. then, recovering and purifying a PCR product by using a glue recovery kit;

c. finally, cloning and transforming, screening positive clone sub-colonies, and sequencing after amplification culture;

example two, the rate of inhibition of Paenibacillus polymyxa HJ-2 against different Fusarium was tested

(1) Inoculating F.solani f.sp.paris, F.graminearum f.sp.paris, F.oxysporum, F.equiseti and F.tricinctum cakes in the center of a PDA solid culture medium plate, placing a sterile filter paper round cake with the diameter of 6mm at the position of 3.0cm on one side of the cake, inoculating a paenibacillus polymyxa strain HJ-2 to the sterile filter paper, adding a blank control on the other side of the cake, inoculating sterile filter paper to sterile water, repeating the steps for 5 times for each test strain, and placing the test strain in an incubator at 25 ℃ for dark culture;

(2) observing and measuring the width of the bacteriostatic band after 4-8 days according to the growth rate of pathogenic bacteria, and measuring the bacteriostatic rate, wherein the specific results are shown in table 2;

table 2: bacteriostasis rate of paenibacillus polymyxa HJ-2 on different fusarium

The bacteriostasis rate is (radius of colony at the control side-radius of colony at the opposite side)/radius of colony at the control side is 100%;

as is clear from the results of the examination in table 2, paenibacillus polymyxa HJ-2 of the present invention has an inhibitory effect on root rot of paris polyphylla f.solani f.sp.paris, f.graminearum f.sp.paris, f.oxysporum, f.equiseti, f.tricinctum.

In the third embodiment, the influence of the fermentation filtrate of the paenibacillus polymyxa strain HJ-2 on the activity of fusarium F. oxysporum zoospores is detected, and the specific steps are as follows:

the method comprises the following steps: activating the strain HJ-2, inoculating into 250mL triangular flask containing 50mL fermentation medium, shake culturing at 28 deg.C and 150rpm for 2 days to make the concentration of the bacterial liquid reach 2.0 × 10⁹cfu/ml, centrifuging the fermentation liquid at 12000rpm for 5min, and filtering with 0.22 μm bacterial filter to obtain sterile fermentation filtrate;

step two: three groups of experiments are divided, one group takes 450 mu l zoospore and 450 mu l sterile fermentation filtrate, the other group takes 450 mu l zoospore and 450 mu l fermentation filtrate, the zoospore and 450 mu l sterile water suspension are taken as a control, the zoospore and 450 mu l sterile water suspension are placed at 25 ℃, microscopic observation is carried out every 3 hours, and when the germination rate of the control is 90%, the germination rates of other two treated fusarium F. oxysporum spores are counted;

the inhibition rate (%) of spore germination was ═ control germination rate-treatment germination rate)/control germination rate × 100%

The effect of the fermentation filtrate of paenibacillus polymyxa strain HJ-2 on the activity of fusarium f.oxysporum zoospores is shown in table 3;

table 3: effect of Paenibacillus polymyxa HJ-2 on Fusarium F

The experimental results of table 3 show that: zoospore in the control group normally germinates under a microscope, zoospore in the treatment group shows no germination, short germinated germ tube and the like, and then deformity with different degrees, such as enlargement or vesicle shape, is found; finally, the spores are largely broken, and the germination quantity is obviously reduced.

The fourth embodiment of the invention provides an experiment for detecting the control effect of paenibacillus polymyxa strain HJ-2 on the root rot of paris polyphylla, which comprises the following specific steps:

the method comprises the following steps: the concentration obtained in the first two steps of the example is 2.0X 10⁸cfu/mL-5.0×10⁸cfu/mL liquid microbial inoculum, adopting root irrigation method to prevent and control root rot of Paris polyphylla at the early stage of emergence of Paris polyphylla, wherein the dosage is microbial inoculum stock solution according to 500mL/m²Setting two groups of experiments, taking six plants of paris polyphylla growing in a nutrition pot and having similar growth vigor, performing root irrigation treatment on one group of paris polyphylla with clear water and growing naturally, performing root irrigation treatment on the other group of paris polyphylla, performing root irrigation once in 7 days, and continuously preventing and treating for 3 times; meanwhile, two communities are arranged in the field, one group of the communities is subjected to root irrigation treatment by clear water and naturally grows, the other group is subjected to root irrigation treatment, the roots are irrigated once in 7 days, and the continuous control is performed for 3 times;

step two: investigating the occurrence condition of the root rot of Paris polyphylla in a residential area in late 7 th ten days, calculating the prevention and treatment effect, and using clear water to irrigate roots as a control;

incidence (%) ═ number of diseased plants/total number of investigated plants × 100%;

control effect (%) ═ control incidence-treatment incidence)/control incidence × 100%;

the control effect investigation is shown in table 4;

table 4: prevention and treatment effect of bacillus strain HJ-2 on root rot of paris polyphylla

The experimental results of table 4 show that: after the root of Paris polyphylla is irrigated by using the Paenibacillus polymyxa strain HJ-2, the severity and disease index of the disease of Paris polyphylla are obviously reduced, and the relative prevention effect and the disease alleviation degree of the Paris polyphylla are obviously improved, so that the effect of HJ-2 on preventing the root rot of Paris polyphylla is obvious; wherein: FIG. 5(a) root irrigation experiment (Nutrition bowl, control); FIG. 5(b) root irrigation experiment (nutrition pot, HJ-2); fig. 5(c) root irrigation experiment (plot, control); FIG. 5(d) root irrigation experiment (nutrition pot, HJ-2).

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims (2)

1. The bacillus is Paenibacillus polymyxa HJ-2, is separated from rhizosphere soil of Paris polyphylla in Shibata, Hubei, and is preserved in China center for type culture Collection CCTCC (China center for type culture Collection) in Wuhan university, Wuhan, Hubei, with the preservation number of CCTCC number M2019617.

2. The application of the paenibacillus polymyxa HJ-2 in preventing and treating the root rot of paris polyphylla as claimed in claim 1, wherein the concrete steps of the application of the bacillus are as follows:

the method comprises the following steps: activating the strain HJ-2, inoculating into 250mL triangular flask containing 50mL fermentation medium, shake culturing at 28 deg.C and 150rpm for 2 days to make the concentration of the bacterial liquid reach 2.0 × 10⁸cfu／ml—5.0×10⁸cfu/mL to obtain a liquid microbial inoculum;

step two: the concentration obtained in the first step is 2.0 multiplied by 10⁸cfu/mL—5.0×10⁸The cfu/mL liquid microbial inoculum adopts a root irrigation method to prevent and control the root rot of the paris polyphylla at the early emergence stage of the paris polyphylla, and the root irrigation is carried out once in 7 days for 3 times continuously.

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