CN114933982B

CN114933982B - Bacillus belicus and application thereof in preventing and treating sweet potato stem root rot

Info

Publication number: CN114933982B
Application number: CN202210307165.8A
Authority: CN
Inventors: 朱丽燕; 何晓婵; 朱浩; 周小军; 李由; 黄鑫; 陈忠友; 陈晓; 马赵江; 朱强兴
Original assignee: Zhejiang Agricultural Machinery Research Institute
Current assignee: Zhejiang Agricultural Machinery Research Institute
Priority date: 2022-03-25
Filing date: 2022-03-25
Publication date: 2024-04-19
Anticipated expiration: 2042-03-25
Also published as: CN114933982A

Abstract

The invention discloses bacillus beleiensis and application thereof in preventing and treating sweet potato stem root rot, wherein the bacillus beleiensis is classified and named Bacillus velezensis, the strain number is JH22, and the preservation number is: cctccc NO: m2022299. The bacillus belicus Bacillus velezensis JH screened by the invention has high-efficiency antagonism to the dadan dieldrin bacteria, can be used for preventing and controlling sweet potato stem root rot caused by the dadan dieldrin bacteria, and has a greenhouse potting test prevention effect of 54.1%. The strain has ideal control effect in the early stage of plant disease, and the characteristic shows that the strain has great application potential as a biological prevention pesticide.

Description

Bacillus belicus and application thereof in preventing and treating sweet potato stem root rot

Technical Field

The invention relates to the technical fields of development and utilization of microbial germplasm resources and biological control of plant diseases, in particular to application of bacillus belicus for controlling sweet potato stem root rot and a sweet potato cultivation method.

Background

Sweet potatoes are staple food crops with important strategic significance and taking tubers as food, and are widely planted worldwide. Currently, asia is the world's second big sweet potato growing continent, china is the world's first big sweet potato producing country, and chinese sweet potato yield is about 56.6% of the world's total yield. Sweet potato contains abundant carbohydrate, high-quality protein, dietary fiber, carotene, and microelements such as potassium, zinc, calcium, and iron, and can provide important nutrients for human body and energy for biological activity.

Sweet potato stem root rot (stem and root rot) is a bacterial disease caused by dadantikok bacteria (DICKEYA DADANTII) of the genus dickeya, originally known as erwinia chrysanthemi ERWINIA CHRYSANTHEMI. Disease seriously affects the yield and quality of sweet potato. The common incidence rate of field diseases is between 10 and 20 percent, and the incidence rate of serious field diseases reaches more than 50 percent, and the field diseases are in serious condition to be in dead harvest. The disease constitutes a great threat to the sweet potato production and food safety of sweet potato producing areas such as Zhejiang province, guangdong province, jiangsu province, shandong province and the like in China. The pathogenic bacteria has wide host range, and is a harmful plant of Convolvulaceae, compositae and Orchidaceae, and vegetable plant of Solanaceae, leguminosae, gramineae, and Brassicaceae, etc., and 60 kinds of plants including sweet potato, flos Chrysanthemi, tobacco, fructus Piperis, tomato, potato, cabbage, eggplant, soybean, morning glory, semen Cuscutae, etc.

Pathogenic bacteria invade mainly through the wound of the host, producing pectic enzymes to degrade pectin in the plant cell wall, thereby causing the vascular bundles to be hollow and rotten, causing the stalks to be black brown, the plants to wilt and have a rancid odor. Although pathogenic bacteria cannot survive in soil for long periods of time, they can survive around plant debris, weeds, or the roots of other plants. In addition, the primary source of infection may include disease potatoes, vines, equipment contaminated during intercropping operations, and the like.

Because of the wide range of pathogenic bacteria hosts and diversity of populations of the sweet potato stem root rot, no effective prevention and control method for preventing and controlling the stem root rot exists at present. As the sweet potato cultivation area is still limited relative to crops, the disease and disease-resistant breeding aspects are obviously not researched enough, and no disease-resistant variety is available in production. Disease control mainly depends on bactericides such as common bacterial bactericides including 0.3% tetramycin, 72% agricultural streptomycin, 6% kasugamycin and the like. The widely used bactericides not only kill beneficial microorganisms in soil, but also cause the generation of drug resistance of pathogenic bacteria, and the use of a large amount of bactericides has potential harm to ecology and human health. In recent years, a great deal of research at home and abroad shows that the development of an anti-microbial agent by utilizing antagonistic microorganisms has become a necessary trend of development, such as biological control of plant diseases such as phytophthora capsici, sclerotium disease of rape, wheat scab and rice blast by using bacteria such as trichoderma fungi, pseudomonas, bacillus and the like. Sweet potato stem root rot is a destructive disease, and after pathogenic bacteria infection, the disease is rapidly developed, and serious economic loss can be caused in a short period of time. However, the reports of preventing and controlling the root rot of sweet potato by using biological prevention and control means at home and abroad are relatively limited at present. In order to effectively prevent and treat the serious occurrence of the disease, biological prevention and treatment research of the disease is developed, and disease grading standards are established according to field and indoor disease research; screening biocontrol strains and researching antagonistic mechanisms thereof; and a disease control test is carried out, so that a reliable theoretical basis is provided for the application of biocontrol strains. And the development and application of biocontrol bacteria can provide wide market and prospect for preventing and treating sweet potato stem root rot.

Disclosure of Invention

The invention provides bacillus belicus which can be used for preventing and treating sweet potato stem root rot, and the screened strain has high-efficiency antagonism on dadan Dike bacteria, can be used for preventing and treating sweet potato stem root rot caused by dadan Dike bacteria, and aims to provide a new thought and a new means for solving the sweet potato stem root rot.

Bacillus belicus, classified and named Bacillus velezensis, strain No. JH22, was preserved in China center for type culture Collection, with a preservation number of CCTCC NO: m2022299.

Biological and morphological characteristics of the strain:

The strain JH22 is cultivated on an NA flat plate at constant temperature (30 ℃) for 24 hours, bacterial colonies are irregular round, the bacterial colonies are easy to accumulate into a line shape, the bacterial colonies are milky white and opaque, the surface is dry, the bacteria inoculating ring is slightly sticky when being light touched, and the edge of the bacteria inoculating ring is provided with folds.

Genetic characteristics of the strain:

the 16S rRNA sequence of the strain JH22 is shown as SEQ ID NO: 13; the gyrA gene sequence is shown as SEQ ID NO: 14. shown; the rpoB gene sequence is shown in SEQ ID NO: 15; the purH gene sequence is shown in SEQ ID NO: shown at 16; the groEL gene sequence is shown in SEQ ID NO: shown at 17; polC gene sequence is shown in SEQ ID NO: shown at 18.

The invention also provides application of the bacillus belicus in antagonizing pathogenic bacteria dactylotheca (DICKEYA DADANTII). Such as for the preparation of a bacterial agent against the pathogenic bacterium Deuteria dadanti (DICKEYA DADANTII) ZJ 97.

The invention also provides application of the bacillus belicus in preventing and treating sweet potato stem root rot.

The invention also provides application of the bacillus belicus in promoting sweet potato growth.

Alternatively, a microbial inoculum comprising the bacillus belicus is applied to the root or stem base of sweet potato seedlings.

The invention also provides a microbial inoculum for preventing and treating sweet potato stem root rot, which comprises the bacillus belicus.

Alternatively, the Bacillus belicus concentration is not less than 10 ⁷ CFU/mL, preferably about 10 ⁷ CFU/mL.

The invention also provides a sweet potato biological control method or a sweet potato growth promoting method: selecting cutting potato seedlings with three to four sections of lengths, and irrigating the root parts of the potato seedlings with a microbial inoculum containing the bacillus beljalis.

Optionally, the concentration of bacillus beliensis in the microbial inoculum is not lower than 10 ⁷ CFU/mL.

Further optionally, the concentration of bacillus beliensis in the microbial inoculum is about 10 ⁷ CFU/mL; watering the microbial inoculum once again at intervals of 10-15 days; the irrigation amount is 5-15 mL (preferably 10 mL) per plant.

Compared with the prior art, the invention has at least one of the following beneficial effects:

(1) The bacillus belicus Bacillus velezensis JH screened by the invention has high-efficiency antagonism to the dadan dieldrin bacteria, can be used for preventing and controlling sweet potato stem root rot caused by the dadan dieldrin bacteria, and has a greenhouse potting test prevention effect of 54.1 percent through a conventional bacteria injured inoculation prevention and control test. The strain has ideal control effect in the early stage of plant disease, and the characteristic shows that the strain has great application potential as a biological prevention microbial preparation;

(2) The bacillus belicus Bacillus velezensis JH screened by the invention has remarkable inhibition effect on the growth of pathogenic bacteria of the dadan Dikkera, the inhibition rate reaches 75.8 percent, and the inhibition effect of the sterile cell supernatant liquid of the bacillus belicus Bacillus velezensis JH on the growth of the pathogenic bacteria reaches 68.8 percent;

(3) The control effect of the bacillus belicus Bacillus velezensis JH screened by the invention on the sweet potato with wound pathogen infection condition is 54.1%;

(4) The bacillus belicus Bacillus velezensis JH antagonistic compound screened by the invention has high expression quantity;

(5) The bacillus belicus Bacillus velezensis JH screened by the method has good growth promoting effect on sweet potatoes.

Drawings

FIG. 1 is a graph showing the in vitro inhibition of Klebsiella diminuta by 5 biocontrol bacteria (A: strain JH22, B: strain JH38, C: strain JH59, D: strain JH35, E: strain JH 16);

FIG. 2 is a diagram showing morphology observation of strain JH22 on NA plates;

FIG. 3 is a phylogenetic tree constructed by combining the gene sequences of the gyrA gene, the rpoB gene, the purH gene, the groEL gene and polC of Bacillus bailii;

FIG. 4 shows MALDI-TOF-MS peak patterns of strain JH22 and JH38 lipopeptides (A: JH22, B: JH 38);

FIG. 5 is a graph showing the effect of strain JH22 on biofilm formation by the pathogenic bacteria Dekkera dadans (A: color change in wells of different dilution factors, B: percent inhibition of biofilm formation);

FIG. 6 is a graph showing the effect of strain JH22 on the motility of the pathogenic bacteria Dekkera dadans;

FIG. 7 is a graph showing the effect of strain JH22 on the colonisation movement by the pathogenic bacteria Dekkera dadans;

FIG. 8 is a graph showing the effect of the strain JH22 on preventing and controlling root rot of sweet potato in a greenhouse, wherein the graph A is a control, and the graph B is a treatment group;

FIG. 9 is a graph showing the results of the plant JH22 on the sweet potato's pro-effect (left panel, right panel).

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The soil environment is complex and various, the effect of biocontrol diseases usually depends on the local adaptability of the biocontrol agent to soil, and the introduced strain has poor competitiveness and poor soil adaptability and can only have limited success. The screening of high-efficiency biocontrol strains for different pathogenic bacteria in different areas is still the direction of attention of scientific researchers in various countries at present or even in the future. Therefore, the invention screens out the high-efficiency biocontrol strain suitable for the soil environment of the place, and performs related prevention and treatment research work according to the local conditions, which is an important task for the biological prevention and treatment of the diseases.

The biological control antagonistic strains for the sweet potato stem root rot are screened, so that the sweet potato stem root rot can be effectively controlled, the disease control cost is reduced, the sweet potato yield is improved, the environment is protected, the biological safety is realized, and the promotion effect is provided for the industrial production of products. Bacillus is an important member of bacterial biocontrol, can be used as biocontrol agent to replace chemical pesticides in partial agricultural production, and solves the problem of diseases in plant production practice; bacillus also belongs to common plant rooting-promoting bacteria (PGPR) and can produce compounds for promoting plant growth, provide biological nitrogen fixation effect and trigger metabolic activity of plant root systems. The research on bacillus has good sustainable application prospect.

Example 1

1. Strain isolation and screening.

The soil sample to be tested is collected from a continuous cropping sweet potato field with serious disease condition (28 DEG 48 '14' N,119 DEG 20 '56' E) in Jin Huashi wu urban area of Zhejiang province in 12 months in 2020, the soil sample near the root system of the disease plant is randomly collected in a sterile bag, sealed and stored in a refrigerator at 4 ℃.

NA plates screen for biocontrol bacteria in soil: 1g of soil sample is taken in sterile water and is oscillated for 3min to prepare soil suspension, and the dilution concentration is 10 ^-4～10^-6 gradient. The prepared soil suspension was uniformly coated on an NA plate with a sterile coating rod and grown at 30℃for 48 hours. According to the shape, color, size, regularity, convexity and other characters of the colony, selecting different single colonies, purifying each colony for 3 times, preparing bacterial liquid, adding 30% glycerol and storing in a freezer at-40 ℃. The 15 soil samples were screened for 301 strains altogether.

2. And (5) screening biocontrol strains.

Antagonistic activity of the isolated strain was evaluated by an agar diffusion method. All the isolated strains were inoculated into a liquid NA medium and cultured in a HZQ-F100 shaker at 30℃and 200rpm for 24 hours to obtain a bacterial liquid (adjusted to about 10 ⁷ CFU/mL). Similarly, the pathogenic organism, dactylotheca tumefaciens ZJ97, was also inoculated into the liquid NA medium and cultured under the above conditions. 400 μl of the Dekkera dadans bacterial solution (10 ⁸ CFU/mL) was applied to the surface of the NA plate (diameter 9 cm). 3 equidistant oxford cups (diameter 6 mm) were inserted on the surface, and 25. Mu.l of the strain broth to be screened was added to the oxford cup wells. Each strain was replicated 3 times. After the dishes were placed in a 30℃incubator for 24 hours, the diameter of the zone of inhibition was observed and measured.

According to the diameter of the inhibition zone (diameter is larger than 13.0 mm), 5 candidate strains with high activity (table 1) are obtained through co-screening, and are named as JH16, JH22, JH35, JH38 and JH59 respectively, which obviously inhibit the growth of pathogenic bacteria (P < 0.05), and the inhibition effects are 59.6%, 75.8%, 56.5%, 69.8% and 63.2% respectively.

To determine the activity of the secondary metabolites of the 5 candidate strains, the above-described cultured bacterial suspensions were centrifuged at 6000 rpm for 10 minutes at 4℃respectively, to obtain cell-free supernatants (CFS). And CFS was further filtered through a 0.22- μm filter. Similarly, the pathogen, dactylotheca ZJ97, was inoculated into liquid NA medium, 3 equidistant oxford cups were inserted, and 25 μl of cell-free supernatant was added to oxford cup wells, as described above. After the dishes were placed in a 30℃incubator for 24 hours, the diameter of the zone of inhibition was observed and measured.

The activity measurements of the secondary metabolites of the 5 candidate lines showed that they also significantly inhibited pathogenic bacterial growth (P < 0.05), 55.7%, 68.8%, 52.3%, 62.9%, 56.8%, respectively (table 1). Strains JH22 and JH38 were used for further investigation, since they have a higher inhibitory activity.

TABLE 1 inhibition of biocontrol strains against the pathogenic bacteria Dekkera dadanti

A: bacterial liquid; b: cell-free supernatant

Inhibition (%) = (1-control diameter/treatment group diameter) ×100%

Example 2 identification of strains.

2 Strains screened for high activity were identified.

Morphological observation is shown in FIG. 2, strain JH22 is cultured on NA plate at constant temperature (30 ℃) for 24 hours, the colony is irregular round, the colony is easy to accumulate into a line shape, milky white, opaque, dry on the surface, slightly sticky when the inoculating loop is light touched, and the edge is wrinkled.

To molecular identify the two strains JH22 and JH38, they were subjected to PCR amplification of 16S and 5 housekeeping genes, respectively. The PCR amplification was initiated as shown in Table 2. The PCR amplification reaction system was 50. Mu.L: ddH ₂ O17.5. Mu.L, HLINGENE PCR MASTER Mix 25. Mu.L, 16S-27f 2.5. Mu.L, 16S-1492r 2.5. Mu.L, and DNA template 2.5. Mu.L. The PCR reaction procedure is 5min pre-denaturation at 95 ℃; denaturation at 95℃for 30s; annealing at 56 ℃ for 30s; extending at 72 ℃ for 60 seconds; 35 cycles; preserving at 4 ℃. The PCR amplification product was sequenced bi-directionally by Beijing engine biotechnology Co., ltd (Hangzhou division), wherein the 16S rRNA sequence of strain JH22 is shown in SEQ ID NO: shown at 13. The obtained 16S sequence is aligned with the homologous sequence in GenBank through Blast search engine to determine the corresponding genus.

To distinguish between different closely related species within the genus, sequences of 5 housekeeping genes were used, wherein the gyrA gene sequence of strain JH22 is as shown in SEQ ID NO: 14; the rpoB gene sequence is shown in SEQ ID NO: 15; the purH gene sequence is shown in SEQ ID NO: shown at 16; the groEL gene sequence is shown in SEQ ID NO: shown at 17; polC gene sequence is shown in SEQ ID NO: 18. as shown. And the gyrA gene, rpoB gene, purH gene, groEL gene and polC gene of closely related species were downloaded from GenBank for phylogenetic analysis. And taking the downloaded Bacillus cereus ATCC14579 as an outer group.

To construct the phylogenetic tree, the obtained sequences were compiled with Clustalx1.83, the sequences of each gene were aligned with MAFFT 7.273 software, the ambiguous region was picked with Gblocks 0.91.91 b, the best GTR+I+G nucleotide substitution model was obtained with jModel Test.1.7, and finally the phylogenetic tree was constructed with RaxmlGUI v.1.5 software and with Maximum Likelihood (ML) method.

As shown in FIG. 3, the two test isolates JH22 and JH38 clustered together with Bacillus velezensis BD, BD569, B23190, and B23189 to form a distinct branch with 100% bootstrap support. Thus, both strains were identified as bacillus belicus (Bacillus velezensis). Wherein JH22 is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan at 2022, 3 and 22 days, and the preservation number is CCTCC NO: m2022299.

TABLE 2 PCR primers for amplification

Example 3 detection of lipopeptid compounds from strains.

Lipopeptides of candidate strains are determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bacillus velezensisJH22 and JH38 were inoculated onto NA plates, incubated at 30℃for 24h, and single colonies were picked and dissolved in centrifuge tubes (2.0 mL) containing matrix solution. The matrix solution contained 10mg/mL cyano-4-hydroxycinnamic acid (dissolved in 70% acetonitrile), and 0.1% trifluoroacetic acid (TFA) in 70% acetonitrile. After mixing the solution, it was centrifuged at 5000r/min for 2min, and 1. Mu.L of the sample was then pipetted onto a MALDI-TOF MTP 384 target disk (Germany) and the data recorded using a ultrafleXtreme ^TM MALDI-TOF mass spectrometer (Germany) equipped with a smart beam laser. The measurements were made in a reflective mode of operation and at an ion source acceleration voltage of 20 kV. Mass spectral data are stored in the low mass range region between 0.1 and 2 kD. Lipopeptides produced by these were analyzed according to the lipopeptides series mass spectrum peaks reported and determined in the literature (Table 3).

MALDI-TOF MS analysis showed that lines JH22 and JH38 both produced 3 lipopeptides, including surfactant (surfactins), iturin (iturins) and Fengyin (fengycins) (Table 3). Wherein, the peak (m/z) ranges from 1016 to 1095 as surfactant, from 1030 to 1100 as iturin and from 1450 to 1544 as Fengyuan. In strain JH22, peaks m/z 1030.705 and 1074.723 are assigned to the surfactant; peaks m/z 1043.680, 1057.698, 1065.616, 1079.634 and 1095.619 are ascribed to iturin; peaks m/z 1463.990, 1478.019, 1485.898, 1499.915, 1515.904, 1529.928 and 1543.927 are ascribed to Fengyuan. Similarly, in strain JH38, peaks m/z 1016.691, 1030.706, 1044.725, 1058.741 and 1074.723 are ascribed to surfactants; peaks m/z 1065.595 and 1079.613 are ascribed to iturin; peaks m/z 1471.946, 1485.885, 1499.901, 1513.585 and 1529.811 are ascribed to Fengyuan.

Although both lines JH22 and JH38 produced 3 lipopeptides, line JH22 produced 3 lipopeptides significantly more strongly than JH38 (FIG. 4), indicating that line JH22 produced more lipopeptides than line JH38, which is also consistent with the in vitro bioactivity test results described above. Thus, strain JH22 was selected as the most potential biocontrol strain for further investigation of biofilm formation and motility inhibition as well as biocontrol effects and growth promotion on pathogenic bacteria.

Table 3 results of lipopeptide analysis of strains

* : Amino acid at position 7 is Val; * *: the amino acid at position 7 is Leu.

EXAMPLE 4 inhibition of biofilm formation and motility by biocontrol agents

1. Inhibition of biofilm formation.

Preparing biocontrol bacteria seed liquid: and picking Bacillus velezensis JH a single colony, inoculating the single colony into 10mL of liquid NA (agar-free) liquid culture medium, and shake-culturing for 24 hours at 30 ℃ and 200rpm until the OD ₆₀₀ of the culture solution is 0.6-0.8, thereby obtaining the liquid fermentation seed liquid of the biocontrol agent.

Liquid fermentation: taking 500 mu L of biocontrol bacteria seed liquid, inoculating the biocontrol bacteria seed liquid into 500mL of liquid NA (agar-free) culture medium, and carrying out shake culture for 60h at 30 ℃ and 200rpm to obtain biocontrol bacteria liquid fermentation liquid. And (3) regulating the concentration of bacillus beljalis in the fermentation liquid to about 10 ⁷ CFU/mL to obtain the biocontrol microbial agent.

Preparation of cell-free supernatant: centrifuging the biocontrol bacterial liquid fermentation broth at 6000rpm for 10min, and filtering residual thalli by using a 0.22 mu m bacterial filter to obtain Bacillus velezensis JH cell-free supernatant.

To determine the effect of biocontrol-cell-free supernatants on biofilm formation by pathogenic bacteria, biofilm formation inhibition assays were performed in 96-well plates. First, 40. Mu.l of 5, 10, 25, 50 and 100 fold diluted cell-free supernatant was added to each well, followed by 20. Mu.l of the pathogenic bacteria Dardalidi bacteria solution (10 ⁸ CFU/mL) and 140. Mu.l of liquid NA (agar-free) medium. Finally, the volume in each plate well was brought to 200 μl, and the cell-free supernatant was finally diluted to 25, 50, 125, 250 and 500 times, respectively. And each treatment was repeated 3 times with the addition of 40. Mu.l of sterile physiological saline as a control. After the 96-well plate was left to stand at 30℃for 24 hours, the culture solution was poured off, washed 3 times with sterilized water, the non-adherent cells were removed, air-dried in a super clean bench and then 100. Mu.l of 99% methanol was added to each well for 15 minutes, to fix the biofilm. After removal of methanol, staining with 1% Crystal Violet (CV) solution for 30min, removal of free staining, washing with deionized water twice, and then eluting the biofilm with 200 μl 33% acetic acid, the solution was transferred to a clean 96-well plate to quantify the amount of biofilm formation. Quantification of biofilm formation was determined using a Lambda 35UV/VIS (USA) spectrophotometer at 590nm (OD ₅₉₀).

The biofilm inhibition test results showed (fig. 5 a) that the change in color from light purple to dark purple in the wells was corresponding to an increase in dilution concentration (25, 50, 125, 250 and 500 fold), i.e., an increase in biofilm formation. Depending on the percentage of biofilm inhibition, the cell-free supernatant was the most inhibitory activity on biofilm formation by 25-fold dilution (B in fig. 5), followed by 50, 125, 250 and 500-fold cell-free supernatant dilutions, respectively. In addition, there was no significant difference between 250-fold and 500-fold cell-free supernatant dilutions (P < 0.05).

2. Pathogen motility inhibition assay.

To determine the inhibition of pathogen motility by cell-free fermentation broth produced by Bacillus velezensis JH, a motility inhibition assay was performed on semi-solid SM medium (3 g/L beef extract, 5g/L peptone, agar of varying concentrations and 20% glucose 25 ml/L). To determine the motility inhibiting effect of the cell-free fermentation broth on the pathogenic bacteria dactylotheca, SM medium containing cell-free supernatants 20, 40, 100, 200 and 400 fold, respectively, and 0.3% agar was prepared. Plates without cell-free supernatant served as controls, each treatment was repeated 3 times. After the plates were solidified, 2. Mu.l of a bacterial solution of the pathogenic bacterium Aldrich dirtikola (10 ⁸ CFU/mL) was added to the center of each plate, and the plates were placed in a 30℃incubator and cultured for 12 hours. Then, the colony diameter to the motile of dadant dirac was measured, and the inhibition ratio was calculated. Inhibition (%) = (control colony diameter-treated colony diameter)/control colony diameter x 100%.

The results of the cell-free fermentation broth inhibition effect on the motility of pathogenic bacteria showed that the cell-free fermentation broth treated differently had a significant inhibition effect on the motility of pathogenic bacteria dactylotheca (P < 0.05) (a in fig. 6), and the 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilutions had a significant inhibition effect on the motility of pathogenic bacteria of 92.6%, 88.6%, 81.2%, 68.8% and 62.5%, respectively, but there was no significant difference between the 20-fold and 40-fold dilutions (B in fig. 6).

Similarly, to determine the inhibitory effect of cell-free fermentation broths on the colonization by the pathogenic bacteria dadantikok, SM medium containing cell-free supernatants 20, 40, 100, 200 and 400 fold and 0.5% agar was prepared. Plates without cell-free supernatant served as controls, each treatment was repeated 3 times. After the plates were solidified, 2. Mu.l of a bacterial solution of the pathogenic bacterium Aldrich dirtikola (10 ⁸ CFU/mL) was added to the center of each plate, and the plates were placed in a 30℃incubator and cultured for 12 hours. Colony diameters swimming to dactylotheca were measured and inhibition rates were calculated as above.

The results of the cell-free fermentation broth inhibition effect on the aggregation of pathogenic bacteria show that the cell-free fermentation broth treated in different ways has a significant inhibition effect on the aggregation of pathogenic bacteria dactylotheca (P < 0.05) (A in FIG. 7), and the 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilutions have a significant inhibition effect on the motility of pathogenic bacteria of 93.4%, 90.4%, 81.1%, 73.2% and 64.0%, respectively, but there is no significant difference between the 20-fold and 40-fold dilutions (B in FIG. 7)

The pathogenic bacteria dadant direc bacteria form a biological film after reaching the surface of a host through swimming and clustering movement under the stimulation of host secretion, and the generation of the biological film promotes the interaction of the pathogenic bacteria and the host and then infects the host. The research provides Bacillus velezensis JH that can inhibit the mobility, the cluster movement and the formation of biological films of pathogenic bacteria, and provides another theoretical basis for the strain as biocontrol bacteria.

Example 5

1. Greenhouse experiments for biocontrol and prevention of sweet potato stem root rot.

For measuring the control effect of the biocontrol strain in vivo, bacillus velezensis JH strain is selected for a greenhouse disease control test. Selecting healthy sweet potato (Zhejiang potato No. 13, a disease-sensitive variety) and sterilizing with 75% alcohol for 5min, and washing with sterile water. And then sterilizing the surface of the potato block for 5min by using a 2% sodium hypochlorite solution, flushing the potato block with sterile water for 3 times, and then air-drying the potato block in an ultra-clean workbench. The potato blocks are sown in a vegetable cultivation plastic basket containing nutrient soil. The basket is then moved into a glass greenhouse at a temperature of 25-28 ℃ and a relative humidity of 80-90%. After 30 days (4-5 leaf period), selecting seedlings with consistent height, cutting each seedling from potato blocks (the seedling base contains a small amount of potato block residues), and transplanting the seedling into a new flowerpot containing nutrient soil. Each seedling stem base was pricked with a sterile needle, 10mL (10 ⁷ CFU/mL) of the prepared Bacillus bailii microbial inoculum was irrigated to each sweet potato seedling stem base, and 10mL of sterilized water was inoculated as a control. And after 24 hours, 10mL (10 ⁷ CFU/mL) of the prepared pathogenic bacteria of the Dekkera dadanti bacteria are respectively irrigated on the base of each seedling stem. Each treatment was repeated 3 times for 30 plants under the same culture conditions. After the disease occurs, the disease occurrence symptoms are observed every day, and after 20 days, the disease incidence, disease severity and prevention and treatment effects are counted. Disease grading criteria are as follows (using a scale of 0 to 4):

0 = healthy, asymptomatic;

1 = healthy aerial plants, only around the basal part of the ground stem with visible brown symptoms;

2 = stem basal blackish brown symptoms are evident, and the upper part of the plant has yellow leaves or curled leaves or sagged leaves;

3=the leaves of the plants on the ground are seriously yellowing or the plants are withered, so that the stem base is seriously rotten;

4 = whole plant wither or die.

Incidence (%) = number of diseased plants/total number of plants x 100%;

disease index (%) =Σ (grade value×number of plants)/(4×total number of plants) ×100%;

Control effect (%) = (control disease index-treatment disease index)/control disease index x 100%.

The greenhouse experimental results of biocontrol and prevention of sweet potato stem root rot show that (figure 8) the biocontrol microbial inoculum can delay leaf symptoms and obviously reduce the incidence and severity of sweet potato stem diseases. The control group inoculated with pathogenic bacteria showed harmful symptoms 57 days after inoculation, dark brown symptoms near the stem base of the soil line, while the treated group plants irrigated with the microbial inoculum had no disease symptoms. 11 days after inoculation, the treated (watered) part of the plants showed similar harmful symptoms, while the control part showed serious disease symptoms. After 20 days, most of the leaves of the plants treated by the control treatment turn yellow, the rot symptoms of stems and roots are serious, individual plants are withered or dead (A in figure 8), and the inoculated biocontrol bacteria have no wither symptoms, only individual leaves turn yellow (B in figure 8), and a good biocontrol effect is shown. Statistical results of disease control show (table 4), the biological control reduces disease incidence by 37%, disease index by 27% and control effect by 54.1%. At the same time, it was also observed that the biocontrol microbial agents significantly promote plant growth.

TABLE 4 greenhouse control effect of biocontrol microbial inoculum on sweet potato stem root rot

2. Growth promoting effect of biocontrol strain on sweet potato growth.

To evaluate the Plant Growth Promoting (PGP) ability of Bacillus velezensis JH microbial agents, PGP experiments were performed under greenhouse conditions (conditions as above). The cultivation method of Zhejiang potato No. 13 seedling is the same as above. After 30 days of greenhouse cultivation, sweet potato seedlings with the length and the size similar to each other are selected, stolen from the base of the stem and cultivated into a pot containing nutrient soil. After planting, 10mL Bacillus velezensis JH of the inoculum (about 10 ⁷ CFU/mL) was irrigated. The preparation method of the microbial inoculum is the same as that of the microbial inoculum. 10mL of microbial inoculum is irrigated once again after 10-15 days. The same volume of sterilized water was irrigated as a control. Every 30 sweet potato seedlings were treated as one treatment, and each treatment was repeated 3 times. After 45 days of cultivation, the pro-active effect was counted. To determine the dry weight of the plant organs, they were placed in an oven and dried for 3 days at 65 ℃. The pro-effects were evaluated by percent difference, percent difference = (treatment-control)/control x 100%.

The results of the pro-efficacy tests showed that the biocontrol microbial agents significantly promoted an increase in plant biomass (fig. 9), and increased the stem length, root length, seedling fresh weight, seedling dry weight, root fresh weight and root dry weight of sweet potato by 30.3%, 28.8%, 21.1%, 49.7%, 54.7% and 65.0%, respectively, after 45 days (table 5).

TABLE 5 Protoffee of biocontrol microbial agent on sweet potato

Aiming at the current situation that the root rot of the sweet potato is difficult to prevent and treat, the invention screens out the bacillus belicus strain JH22 which can effectively prevent and treat the root rot of the sweet potato caused by pathogenic bacteria Daden Dike bacteria, and the biocontrol agent can effectively inhibit the formation of the biological film of the Daden Dike bacteria and the mobility and the clustering movement of thalli, and the biocontrol agent or biological pesticide developed by using the strain has good application prospect.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

SEQUENCE LISTING

<110> Jinhua city agricultural science institute (Zhejiang province agricultural machinery institute)

<120> Bacillus belicus and application thereof in preventing and treating sweet potato stem root rot

<130>

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 2

aaggaggtga tccagccgca 20

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 3

cagtcaggaa atgcgtacgt cctt 24

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 4

caaggtaatg ctccaggcat tgct 24

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 5

gacgtgggat ggctacaact 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 6

attgtcgcct ttaacgatgg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 7

acagagcttg gcgttgaagt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 8

gcttcttggc tgaatgaagg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 9

ttgtcgctca yaatgcaagc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 10

ytcaagcatt tcrtctgtcg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 11

gagcttgaag tkgttgaagg 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 12

tgagcgtgtw acttttgtwg 20

<210> 13

<211> 1440

<212> DNA

<213> Bacillus velezensis

<400> 13

aaagctactt cggcggctgg ctcctaaagg ttacctcacc gacttcgggt gttacaaact 60

ctcgtggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga 120

tccgcgatta ctagcgattc cagcttcacg cagtcgagtt gcagactgcg atccgaactg 180

agaacagatt tgtgggattg gcttaacctc gcggtttcgc tgccctttgt tctgtccatt 240

gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc 300

ctccggtttg tcaccggcag tcaccttaga gtgcccaact gaatgctggc aactaagatc 360

aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc 420

atgcaccacc tgtcactctg cccccgaagg ggacgtccta tctctaggat tgtcagagga 480

tgtcaagacc tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt 540

gtgcgggccc ccgtcaattc ctttgagttt cagtcttgcg accgtactcc ccaggcggag 600

tgcttaatgc gttagctgca gcactaaggg gcggaaaccc cctaacactt agcactcatc 660

gtttacggcg tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgctcctc 720

agcgtcagtt acagaccaga gagtcgcctt cgccactggt gttcctccac atctctacgc 780

atttcaccgc tacacgtgga attccactct cctcttctgc actcaagttc cccagtttcc 840

aatgaccctc cccggttgag ccgggggctt tcacatcaga cttaagaaac cgcctgcgag 900

ccctttacgc ccaataattc cggacaacgc ttgccaccta cgtattaccg cggctgctgg 960

cacgtagtta gccgtggctt tctggttagg taccgtcaag gtgccgccct atttgaacgg 1020

cacttgttct tccctaacaa cagagcttta cgatccgaaa accttcatca ctcacgcggc 1080

gttgctccgt cagactttcg tccattgcgg aagattccct actgctgcct cccgtaggag 1140

tctgggccgt gtctcagtcc cagtgtggcc gatcaccctc tcaggtcggc tacgcatcgt 1200

cgccttggtg agccgttacc tcaccaacta gctaatgcgc cgcgggtcca tctgtaagtg 1260

gtagccgaag ccacctttta tgtctgaacc atgcggttca gacaaccatc cggtattagc 1320

cccggtttcc cggagttatc ccagtcttac aggcaggtta cccacgtgtt actcacccgt 1380

ccgccgctaa catcagggag caagctccca tctgtccgct cgactgcatt atagcagccg 1440

<210> 14

<211> 960

<212> DNA

<213> Bacillus velezensis

<400> 14

gacgtatgca gatgagcgtt atcgtatccc gggcgcttcc ggatgtgcgt gacggtctga 60

agccggttca cagacggatt ttgtacgcaa tgaatgattt aggcatgacc agtgacaaac 120

catataaaaa atctgcccgt atcgtcggtg aagttatcgg taagtaccac ccgcacggtg 180

actcagcggt ttacgaatca atggtcagaa tggcgcagga ttttaactac cgctacatgc 240

ttgttgacgg acacggcaac ttcggttcgg ttgacggcga ctcagcggcc gcgatgcgtt 300

acacagaagc gagaatgtca aaaatcgcaa tggaaattct gcgtgacatt acgaaagaca 360

cgattgacta tcaagataac tatgacggtt cagaaagaga gcctgccgtc atgccttcga 420

gatttccgaa tctgctcgta aacggggctg ccggtattgc ggtcggaatg gcgacaaaca 480

ttcccccgca tcagcttggg gaagtcattg aaggcgtgct tgccgtaagt gagaatcctg 540

agattacaaa ccaggagctg atggaataca tcccgggccc ggattttccg actgcaggtc 600

agattttggg ccggagcggc atccgcaagg catatgaatc cggacgggga tcaatcacga 660

tccgggctaa ggctgaaatc gaagagactt catcgggaaa agaaagaatt attgtcacgg 720

aacttcctta tcaggtgaac aaagcgagat taattgaaaa aatcgcggat cttgtccggg 780

acaaaaaaat cgaaggaatt accgatctgc gagacgaatc cgaccgtaac ggaatgagaa 840

tcgtcattga gatccgccgt gacgccaatg ctcacgtcat tttgaataac ctgtacaaac 900

aaacggccct gcagacgtct ttcggaatca acctgctggc gctcgttgac ggacagccga 960

<210> 15

<211> 859

<212> DNA

<213> Bacillus velezensis

<400> 15

gaagcggttc ttgacaatcc ttacatctta atcacagaca aaaaaatcac aaacattcaa 60

gaaatccttc ctgtgcttga gcaagttgta cagcaaggca aaccattgct tctgatcgct 120

gaagatgttg aaggtgaagc tcttgctaca ctcgttgtca acaaacttcg cggcacattc 180

aacgctgttg ccgttaaagc tcctggcttc ggtgaccgcc gtaaagcaat gcttgaagac 240

atctctgttc ttacaggcgg agaagtaatc acagaagact taggccttga cctgaaatct 300

actgaaatcg gacaattggg acgcgcttct aaagttgtgg taacgaaaga aaacacaaca 360

atcgtagaag gcgccggcga cactgaaaaa atcgctgctc gcgtcaacca aatccgcgct 420

caagtggaag aaacaacttc tgaattcgac agagaaaaat tacaagagcg tcttgcgaaa 480

cttgccggcg gcgtagctgt catcaaagtc ggcgctgcga ctgaaactga gctgaaagag 540

cgtaaacttc gcatcgaaga cgccctcaac tcaactcgcg cagctgttga agaaggtatc 600

gtatccggcg gtggtacagc gcttgtcaat gtatacaaca aagtcgctgc agtggaagct 660

gaaggcgatg cgcaaacagg tatcaacatc gtgcttcgcg cgcttgaaga gccgatccgt 720

caaatcgcac acaatgcagg ccttgaagga tctgtcatcg ttgagcgcct gaaaaacgaa 780

aaaatcggcg taggcttcaa cgctgcaacc ggcgaatggg taaacatgat cgaaaaaggt 840

atcgttgacc agacaaaag 859

<210> 16

<211> 818

<212> DNA

<213> Bacillus velezensis

<400> 16

ttgtcgttcc taacgcaagc tttgatatgg gatttttaaa tgtggcgtac aagcgtctac 60

tgaaaacgga aaaagcgaaa aatccggtca ttgatacgct ggaactcgcg cgtttcctgt 120

atcctgagtt taaaaatcac cgcttaaata cgttatgtaa gaagtttgat atcgaattaa 180

cccagcatca ccgagcggtc tttgacgctg aagcaacggg ctacctgctg ttgaaaatgc 240

tcaaagatgc cgctgaaaaa gacatttttt atcatgatca gctgaatgag aatatgggac 300

aatccaatgc ttatcaaaga tcaaggcctt atcacgctac attgcttgcc gtaaatgaga 360

ccggccttaa aaatctgttt aagctcgtgt ccatttctca tattcaatat ttctacagag 420

tgccgcgcat tccgaggtcg cagcttaata aatacagaga aggtctgtta atcggctctg 480

cctgtgacag gggagaggtc tttgaaggca tgatgcaaaa atcacctgaa gaggttgaag 540

atatcgcatc attctatgat tatcttgaag tgcagccgcc ggaagtatac agacaccttc 600

tgcagcttga gctcgtccgg gatgaaaaag cgctgaaaga aatcatcgcc aacatcacga 660

agctcgggga aaaattgaat aagccggtcg ttgccaccgg aaatgtccac tatttaaacg 720

atgaggacaa aatttaccgg aagatcttaa tatcttccca aggcggcgcc aacccgttaa 780

acaggcacga actgcctaaa gtgcacttca gaacgaca 818

<210> 17

<211> 914

<212> DNA

<213> Bacillus velezensis

<400> 17

tcatttcgac cggaggaaca aaaaaacttc ttcaggaaaa cggtgtggat gtcatcggca 60

tttcagaagt gaccggattt cctgaaatta tggacggacg gttaaaaaca ctccatccta 120

atattcacgg cggtctgctt gccgtaagag acaataaaga gcatatggcg cagatcaatg 180

aacacggcat tgcaccgatt gaccttgtgg tcgtcaacct ttatccgttt aaagaaacga 240

tttcaaaaga agacgtaaca tacgatgaag cgatagaaaa cattgatatc ggcggtcccg 300

gcatgctgcg cgccgcatcg aaaaaccatc aggatgtgac ggtcatcaca gaaccggccg 360

attacagctc cgtgctcaat gagatgaaag aacacggcgg cgtttcgctc aaaagaaaac 420

gcgagcttgc ggccaaagta ttccgccata ccgcggcata cgacgcatta atcgctgatt 480

acttaacacg cgaggccggt gagaaagacc ctgagcaatt cactgttact tttgagaaaa 540

aacagtcgct ccgctacggt gaaaaccctc accaagaggc ggttttctac caaagcgcac 600

ttcctgtctc cggttccatc gcagcggcaa aacagcttca cggcaaagag ctttcttata 660

acaatattaa ggacgcggat gcggccgttc aaatcgtccg ggaatttaca gaacccgcag 720

ctgttgccgt taaacatatg aatccatgcg gagtcggtac gggagcttca attgaggaag 780

cattcaataa agcgtatgaa gctgataaaa cctccatttt cggcggcatc atcgcgctga 840

accgtgaagt tgatcaggca acggctgaag cccttcacgg catcttttta aaaatcatta 900

tcgcctcttc tttc 914

<210> 18

<211> 988

<212> DNA

<213> Bacillus velezensis

<400> 18

atcatcatga gtgaacgcct tgtgaaaaga tgatgtatac acatctattc acattgaaga 60

atatgaatca gaagcacgtg atacaaagct tggaccggaa gagatcaccc gcgatattcc 120

aaacgtaggg gaagacgcgc ttcgcaacct tgatgaccgc ggaattatcc gtatcggcgc 180

ggaagtcaac gacggagacc ttctcgtagg taaagtaacg cctaaaggtg taactgagct 240

tacggctgaa gaacgccttc ttcatgcgat ctttggagaa aaagcgcgtg aagtccgtga 300

tacttctctc cgtgtgcctc acggcggcgg cggaattatc cacgacgtaa aagtcttcaa 360

ccgtgaagac ggcgacgaac ttcctccggg agtgaaccag cttgtacgcg tatatatcgt 420

tcagaaacgt aagatttctg aaggtgataa aatggccgga cgtcacggaa ataaaggggt 480

tatctcgaag attcttcctg aagaagatat gccttacctt cctgacggca cgccgatcga 540

tatcatgctt aacccgctgg gtgtaccatc acgtatgaat atcggtcagg tattagaact 600

tcacatgggt atggctgccc gctacctcgg cattcacatc gcgtcacctg tatttgacgg 660

cgcgcgtgaa gaagatgtgt gggaaacact tgaagaagca ggcatgtcaa gagacgctaa 720

aacagttctt tatgacggcc gtacgggaga accgttcgac aaccgtgtat cagtcggaat 780

catgtacatg atcaaactgg ctcacatggt tgacgataaa cttcatgccc gttctacagg 840

tccttactca cttgttacgc agcagcctct cggcggtaaa gcccaattcg gcggacagcg 900

tttcggtgag atggaggttt gggcgcttga agcttacggc gcagcttaca cgcttcaaga 960

aatcctgact gtgaagtccg atgacgtg 988

Claims

1. Bacillus belicus, characterized by the classification designated Bacillus velezensis, strain number JH22, accession number: cctccc NO: m2022299.

2. Use of bacillus belgium according to claim 1 for antagonizing dadant dirac (DICKEYA DADANTII).

3. Use of bacillus belicus according to claim 1 for controlling sweet potato stem root rot.

4. The use of bacillus belgium according to claim 1 for promoting sweet potato growth.

5. The use according to claim 3 or 4, characterized in that the microbial inoculum comprising bacillus beleiensis according to claim 1 is applied to the root or stem base of sweet potato seedlings.

6. A microbial agent for controlling sweet potato stem root rot, comprising bacillus belicus according to claim 1.

7. The microbial agent of claim 6, wherein the concentration of bacillus beljalis is not less than 10 ⁷ CFU/mL.

8. A method for biological control or promotion of sweet potato growth, comprising: selecting a cutting potato seedling with three to four sections of length, and irrigating the root of the potato seedling with a microbial inoculum comprising bacillus belicus according to claim 1; the concentration of bacillus beljalis in the microbial inoculum is not lower than 10 ⁷ CFU/mL; the microbial inoculum is filled again at intervals of 10-15 days; the irrigation amount is 5-15 mL/plant.