CN114933982A

CN114933982A - Bacillus belgii and application thereof in preventing and treating sweet potato stem root rot

Info

Publication number: CN114933982A
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: 2022-08-23
Anticipated expiration: 2042-03-25
Also published as: CN114933982B

Abstract

The invention discloses a Bacillus belgii and application thereof in preventing and treating stem root rot of sweet potatoes, wherein the Bacillus belgii is classified and named as Bacillus velezensis, the strain number is JH22, and the preservation number is as follows: CCTCC NO: m2022299. The Bacillus subtilis JH22 screened by the method has high-efficiency antagonistic action on the Dietzia dadantii, can be used for preventing and treating the stem root rot of the sweet potato caused by the Dietzia dadantii, and has the prevention effect of 54.1 percent in a greenhouse pot culture test. The strain has ideal control effect in the early stage of plant diseases, and the characteristic shows that the strain has great application potential as a biological prevention pesticide.

Description

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

Technical Field

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

Background

Sweet potatoes are staple food crops which have important strategic significance and take tubers as food, and are widely planted in the world. At present, Asia is the second world sweet potato planting continent, China is the first world sweet potato producing country, and the yield of Chinese sweet potatoes accounts for about 56.6 percent of the total world yield. The sweet potato contains abundant carbohydrate, high-quality protein, dietary fiber, carotene, and trace elements such as potassium, zinc, calcium, iron, etc., and can provide important nutrient substances for human body and provide energy for life activities.

Sweet potato stem root rot (stem and root) is a bacterial disease caused by Dieckea dantii (Dickeya dadantii) of the genus Dickeya, originally known as Erwinia chrysanthemi. The disease seriously affects the yield and quality of the sweet potatoes. The disease incidence rate of the field is 10-20% generally, and the disease incidence rate of the field with serious diseases is more than 50%, so that the field is completely harvested under serious conditions. The disease poses a great threat to sweet potato production and food safety in sweet potato production places of Zhejiang province, Guangdong province, Jiangsu province, Shandong province and the like in China. The pathogenic bacteria have wide host range, and are harmful to ornamental plants of Convolvulaceae, Compositae and Orchidaceae and vegetable plants of Solanaceae, Leguminosae, Gramineae and Brassicaceae in 2016 years, and totally more than 60 kinds of plants including sweet potato, flos Chrysanthemi, tobacco, fructus Piperis, fructus Lycopersici Esculenti, rhizoma Solani Tuber osi, caulis et folium Brassicae Capitatae, fructus Solani Melongenae, semen glycines, morning glory, semen Cuscutae, etc.

Pathogenic bacteria mainly invade through wounds of hosts to produce pectinase to degrade pectin in plant cell walls, so that vascular bundles are hollow and rotten, and stem blackbrown, plants are withered and have rancid odor are caused. While pathogenic bacteria do not survive in soil for long periods of time, they can survive around the roots of plant debris, weeds, or other plants. In addition, the initial infection source can comprise diseased potatoes, diseased vines, polluted equipment in the inter-irrigation farming operation and the like.

Because the pathogenic bacteria of the stem and root rot of the sweet potato have wide host range and diversity in population, no effective prevention and control method for preventing and controlling the stem and root rot exists at present. Because the sweet potato cultivation area is still limited compared with crops, the research on diseases and disease-resistant breeding is also insufficient, and no available disease-resistant variety exists in production. Disease control mainly relies on bactericides, such as common bacterial bactericides of 0.3 percent tetramycin, 72 percent agricultural streptomycin, 6 percent kasugamycin and the like. The wide use of the bactericide not only kills beneficial microorganisms in soil, but also causes the generation of drug resistance of pathogenic bacteria, and the use of a large amount of bactericide has potential harm to ecology and human health. In recent years, a great deal of research at home and abroad shows that the research and development of biocontrol agents by utilizing antagonistic microbes becomes a necessary trend for development, for example, the bacteria such as trichoderma fungi, pseudomonas and bacillus are used for biologically preventing and treating plant diseases such as phytophthora capsici leonian, sclerotinia rot of rape, gibberellic disease of wheat and rice blast. The sweet potato stem root rot is a destructive disease, and the disease develops rapidly after being infected by pathogenic bacteria and can cause serious economic loss in a short period of time. However, reports of using biological control means to prevent and treat the sweet potato stem root rot at home and abroad are relatively limited at present. In order to effectively prevent the serious occurrence of the disease, biological prevention and treatment research on the disease is carried out, and a disease classification standard is established according to field and indoor disease research; screening biocontrol strains and researching an antagonistic mechanism of the biocontrol strains; disease control tests are carried out, and reliable theoretical basis is provided for application of the biological control strains. The development and application of the biocontrol bacterium can provide wide market and prospect for controlling the sweet potato stem root rot.

Disclosure of Invention

The invention provides Bacillus belgii for preventing and treating sweet potato stem root rot, the screened strain has high-efficiency antagonistic action on Dietzia dadantii, can be used for preventing and treating the sweet potato stem root rot caused by the Dietzia dadantii, and aims to provide a new idea and a new means for solving the sweet potato stem root rot.

The Bacillus belgii is classified and named as Bacillus velezensis, has the strain number of JH22, is preserved in China center for type culture collection of strains in Wuhan at 3 and 22 months in 2022, and has the preservation number of CCTCC NO: m2022299.

The biological and morphological characteristics of the strain are as follows:

the strain JH22 is cultured on an NA plate for 24h at constant temperature (30 ℃), the bacterial colony is irregular and round, the bacterial colony is easy to accumulate into a linear shape, is milk-white and opaque, has a dry surface, and has slight stickiness and a wrinkled edge when a bacterium inoculating ring is touched.

The genetic characteristics of the strain are as follows:

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

The invention also provides application of the Bacillus belgii in antagonism of pathogenic bacterium Dieckia dadanii (Dickeya dadanii). Such as for the preparation of a bacterial preparation antagonistic to the pathogen Dieckia dadanii (Dickeya dadantii) ZJ 97.

The invention also provides an application of the Bacillus belgii in the prevention and treatment of the stem root rot of the sweet potato.

The invention also provides an application of the Bacillus belgii in sweet potato growth promotion.

Alternatively, a microbial agent comprising the Bacillus belgii is applied to the roots or stem bases of sweet potato seedlings.

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

Optionally, the concentration of Bacillus belgii 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 sweet potato seedlings with the length of three to four sections, and irrigating the roots of the sweet potato seedlings with a microbial inoculum containing the Bacillus beilesiensis.

Optionally, the concentration of the bacillus belgii in the microbial inoculum is not less than 10 ⁷ CFU/mL。

Further optionally, the concentration of Bacillus belgii in the microbial inoculum is about 10 ⁷ CFU/mL; irrigating the microbial inoculum once more every 10 to 15 days; the irrigation amount is 5-15 mL (preferably 10mL) per plant.

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

(1) the screened Bacillus subtilis JH22 has high-efficiency antagonistic action on the Dietzia dadantii, can be used for preventing and treating the stem root rot of the sweet potato caused by the Dietzia dadantii, and has the prevention effect of 54.1 percent through the conventional bacteria wounded inoculation prevention and treatment test and the greenhouse pot culture 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 velezensis JH22 screened by the method has obvious inhibition effect on the growth of pathogenic bacteria Dieckia dadanensis, the inhibition rate reaches 75.8%, and the inhibition effect of the sterile cell supernatant on the growth of pathogenic bacteria reaches 68.8%;

(3) the control effect of the screened Bacillus velezensis JH22 on the infection condition of sweet potato pathogenic bacteria is 54.1%;

(4) the screened Bacillus velezensis JH22 has high expression level of antagonistic compounds;

(5) the Bacillus velezensis JH22 screened by the method has good growth promoting effect on sweet potatoes.

Drawings

FIG. 1 is a graph showing the in vitro inhibitory effect of 5 biocontrol bacteria on Dieckia dadantii (A: strain JH22, B: strain JH38, C: strain JH59, D: strain JH35, E: strain JH 16);

FIG. 2 is a diagram showing the morphological observation of strain JH22 on an NA plate;

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

FIG. 4 is MALDI-TOF-MS peak profiles of strains JH22 and JH38 lipopeptide compounds (A: JH22, B: JH 38);

FIG. 5 is a graph showing the effect of strain JH22 on biofilm formation by the pathogen Dieckia dadantii (A: color change in different dilution fold holes, B: percent inhibition of biofilm formation);

FIG. 6 is a graph showing the effect of strain JH22 on the motility of the pathogenic bacterium Dieckia dadanensis;

FIG. 7 is a graph showing the effect of strain JH22 on colonization by the pathogen Dieckia dadantii;

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

FIG. 9 is a graph showing the results of growth promoting effect of line JH22 on sweetpotato (treatment group on the left and control on the right).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present 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 in the description of the invention herein 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 the soil, and the introduced strains have poor competitiveness and poor soil adaptability and can only achieve limited success. Aiming at different pathogenic bacteria in different areas, screening high-efficiency biocontrol strains is still the direction of attention of researchers in various countries at present and in the future. Therefore, the invention screens out the high-efficiency biocontrol strain suitable for the local soil environment, performs related control research work according to local conditions, and is an important task for biological control of the disease.

Screening the antagonistic strain for biologically controlling the stem and root rot of the sweet potato can effectively control the stem and root rot of the sweet potato, reduce the cost for controlling diseases and improve the yield of the sweet potato on one hand, and can provide a promoting effect on environmental protection, biological safety and industrial production of products on the other hand. The bacillus is an important member for biological control of bacteria, and can be used as a biological control agent to replace chemical pesticides in part of agricultural production, so that the problem of diseases in plant production practice is solved; the bacillus also belongs to common Plant Growth Promoting Rhizobacteria (PGPR), and can produce a compound for promoting plant growth, provide biological nitrogen fixation and trigger the metabolic activity of a plant root system. Has good sustainable application prospect for the research of bacillus.

Example 1

Firstly, strain separation and screening.

The soil samples to be tested were collected from the seriously ill continuous cropping sweet potato fields in the Wuzhou region (28 degrees 48 '14' N, 119 degrees 20 '56' E) in Jinhua City, Zhejiang province in 12 months 2020, and the soil samples near the root systems of the ill plants were randomly collected in sterile bags and stored in a refrigerator at 4 ℃ after sealing.

Screening biocontrol bacteria in soil by using NA plates: taking 1g of soil sample in sterile water, shaking for 3min to prepare a soil suspension, and diluting to 10 ^-4 ～10 ^-6 And (4) gradient. Uniformly coating the prepared soil suspension on an NA flat plate by using a sterile coating rod, and growing at the temperature of 30 DEG CAnd (5) 48 h. Selecting different single colonies according to the shape, color, size, regularity, concave-convex character, etc. of the colonies, purifying each colony for 3 times, preparing bacterial liquid, adding 30% glycerol, and storing in a freezer at-40 deg.C. A total of 301 strains were selected from 15 soil samples.

And secondly, screening the biocontrol strain.

The antagonistic activity of the isolated strains was evaluated by the agar diffusion method. Inoculating all separated strains into liquid NA culture medium, culturing in HZQ-F100 shaker at 30 deg.C and 200rpm for 24 hr to obtain bacterial liquid (adjusted to about 10) ⁷ CFU/mL). Similarly, the pathogenic bacterium Dieckia dadanae ZJ97 was inoculated into the liquid NA medium and cultured under the above conditions. Taking 400 μ l of Dickinsonia dadantii bacterial liquid (10) ⁸ CFU/mL) was coated on the surface of an NA plate (diameter 9 cm). 3 equally spaced oxford cups (diameter 6mm) were inserted into the surface, and 25. mu.l of the fermentation broth of the strain to be screened was added to the holes of the oxford cups. Each strain was replicated 3 times. After the culture dish is placed in an incubator at 30 ℃ for 24 hours, the diameter of the inhibition zone is observed and measured.

According to the diameter of the inhibition zone (the diameter is more than 13.0mm), 5 candidate strains with high activity (table 1) are obtained by co-screening, are named as JH16, JH22, JH35, JH38 and JH59 respectively, and all of the candidate strains can obviously inhibit the growth of pathogenic bacteria (P is less than 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 5 candidate strains, the above-cultured bacterial suspensions were centrifuged at 6000rpm at 4 ℃ for 10 minutes, respectively, to obtain cell-free supernatants (CFS). And the CFS was further filtered through a 0.22- μm filter. Similarly, the pathogen dickinia dadantii ZJ97 was inoculated into liquid NA medium, 3 equally spaced oxford cups were inserted, and 25 μ Ι of cell-free supernatant was added to the oxford cup wells as described above. After the culture dish was placed in an incubator at 30 ℃ for 24 hours, the diameter of the zone of inhibition was observed and measured.

The activity measurement results of the secondary metabolites of 5 candidate strains (shown in figure 1) show that the secondary metabolites also remarkably inhibit the growth of pathogenic bacteria (P <0.05), and the inhibition effects are 55.7%, 68.8%, 52.3%, 62.9% and 56.8% respectively (Table 1). Strains JH22 and JH38 were used for further studies due to their higher inhibitory activity.

TABLE 1 inhibitory Effect of biocontrol strains on the pathogenic bacterium Dieckia dadanii

a: bacterial liquid; b: cell-free supernatant

Inhibition (%) - (1-control diameter/treated group diameter). times.100%

Example 2 identification of strains.

2 strains with high activity are screened out and identified.

The morphological observation is shown in figure 2, the bacterial colony of the strain JH22 is irregularly round after being cultured on an NA plate for 24 hours at constant temperature (30 ℃), the bacterial colony is easily accumulated into a linear shape, is milky white and opaque, has a dry surface, and has a slightly sticky strain receiving ring and a wrinkled edge.

For molecular characterization of the two strains JH22 and JH38, they were subjected to PCR amplification of 16S and 5 housekeeping genes, respectively. The results of PCR amplification are shown in Table 2. The PCR amplification reaction system is 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, DNA template 2.5. mu.L. The PCR reaction program is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 56 ℃ for 30 s; extension at 72 ℃ for 60 s; 35 cycles; storing at 4 ℃. The PCR amplification product was subjected to two-way sequencing by Biotechnology, Inc. of Beijing Ongzhike (Hangzhou division), wherein the 16S rRNA sequence of the strain JH22 is shown as SEQ ID NO: 13, respectively. The obtained 16S sequence is aligned with the homologous sequence in GenBank by a Blast search engine to determine the corresponding genus.

To distinguish between different closely related species within the genus, the sequences of 5 housekeeping genes were used, where the gyrA gene sequence of strain JH22 is shown in SEQ ID NO: 14 is shown in the figure; the rpoB gene sequence is shown as SEQ ID NO: 15 is shown in the figure; the purH gene sequence is shown as SEQ ID NO: 16 is shown in the figure; the groEL gene sequence is shown as SEQ ID NO: 17 is shown; the sequence of the polC gene is shown as SEQ ID NO: 18, respectively. And the gyrA gene, rpoB gene, purH gene, groEL gene and polC gene of closely related species (Table 2) were downloaded from GenBank for phylogenetic analysis. The downloaded Bacillus cereus ATCC14579 was used as the exome.

To construct a phylogenetic tree, the obtained sequences were edited with Clustalx1.83, the sequences of each gene were aligned with MAFFT 7.273 software, ambiguous regions were culled with Gblocks 0.91b, the best GTR + I + G nucleotide substitution model was obtained with jModel Test 2.1.7, and finally a phylogenetic tree was constructed with RaxmlGUI v.1.5 software and with the Maximum Likelihood (ML) method.

As shown in fig. 3, two test isolates JH22 and JH38 clustered together with Bacillus velezensis BD568, BD569, B23190 and B23189 to form a distinct branch with 100% bootlace support. Thus, both strains were identified as Bacillus velezensis. Wherein JH22 has been preserved in China center for type culture Collection in Wuhan at 22.3.2022 with a preservation number of CCTCC NO: m2022299.

TABLE 2 primers for PCR amplification

Example 3 detection of lipopeptide compounds from strains.

The lipopeptide of the candidate strain is determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bacillus velezensis JH22 and JH38 were inoculated onto NA plates, incubated at 30 ℃ for 24h, and single colonies were picked and dissolved in a centrifuge tube (2.0mL) containing a 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. The solution was mixed well and centrifuged at 5000r/min for 2min, then 1. mu.L of the sample was aspirated and dropped on MALDI-TOF MTP 384 target disk (Germany) using ultrafleXtreme equipped with intelligent beam laser ^TM MALDI-TOF mass spectrometer (Germany) recorded the data. The measurements were made in a reflection mode of operation and at an ion source acceleration voltage of 20 kV. Mass spectrum data are stored in the low mass range of 0.1-2 kD. Analyzing according to mass spectrum peak of lipopeptide series reported and determined in literatureThe lipopeptide compounds they produced (table 3).

MALDI-TOF MS analysis showed (FIG. 4) that both strains JH22 and JH38 produced 3 lipopeptide compounds, including surfactin (surfactin), iturin (iturins) and fengycin (fengycins) (Table 3). Wherein the peak (m/z) ranges from 1016 to 1095, 1030 to 1100, and 1450 to 1544, the peak is surfactin, iturin and fengycin. In strain JH22, peaks m/z 1030.705 and 1074.723 are attributed to surfactin; peaks m/z 1043.680, 1057.698, 1065.616, 1079.634 and 1095.619 are attributed to iturin; peaks m/z 1463.990, 1478.019, 1485.898, 1499.915, 1515.904, 1529.928 and 1543.927 are attributed to fengycin. Similarly, in line JH38, peaks m/z 1016.691, 1030.706, 1044.725, 1058.741 and 1074.723 are assigned to surfactin; peaks m/z 1065.595 and 1079.613 are assigned to iturin; peaks m/z 1471.946, 1485.885, 1499.901, 1513.585 and 1529.811 are attributed to fengycin.

While both strains JH22 and JH38 produced 3 lipopeptide compounds, the strength of 3 lipopeptide compounds produced by strain JH22 was significantly higher than that of JH38 (fig. 4), indicating that strain JH22 produced more lipopeptide compounds than strain JH38, which is also consistent with the in vitro bioactivity test results described above. Strain JH22 was therefore selected as the most potent biocontrol strain for further study of biofilm formation and motility inhibition as well as biocontrol and growth promotion in pathogenic bacteria.

TABLE 3 results of lipopeptide analysis of strains

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

Example 4 inhibition of biofilm formation and motility of pathogenic bacteria by biocontrol agents

First, inhibition of biofilm formation.

Preparing biocontrol bacteria seed liquid: a single colony of Bacillus velezensis JH22 was selected and inoculated into 10mL of liquid NA (no agar) liquid medium at 30 ℃ and 200rpmShake-culturing for 24h until culture solution OD ₆₀₀ 0.6-0.8, and is used as a liquid fermentation seed liquid of a biocontrol agent.

Liquid fermentation: inoculating 500 mu L of biocontrol bacteria seed liquid into 500mL of liquid NA (agar-free) culture medium, and performing shake culture at 30 ℃ and 200rpm for 60h to obtain biocontrol bacteria liquid fermentation liquid. Adjusting the concentration of Bacillus belgii in the fermentation broth to about 10 ⁷ CFU/mL to obtain the biocontrol microbial inoculum.

Preparation of cell-free supernatant: and (3) centrifuging the biocontrol bacteria liquid fermentation broth at 6000rpm for 10min, and filtering residual thalli by using a 0.22-micron bacteria filter to obtain the cell-free supernatant of Bacillus velezensis JH 22.

To determine the effect of biocontrol bacteria-free cell supernatants on pathogenic biofilm formation, biofilm formation inhibition assays were performed in 96-well plates. First, 40. mu.l of cell-free supernatant diluted 5, 10, 25, 50 and 100 times was added to each well, and then 20. mu.l of the bacterial strain Dieckia dadaniella (10. mu.l) was added ⁸ CFU/mL), and 140. mu.l of liquid NA (no agar) medium was added. Finally, the volume in each well was brought to 200 μ l, while the cell-free supernatant was finally diluted 25, 50, 125, 250 and 500 fold, 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, and non-adhered cells were removed, air-dried on a clean bench and then 100. mu.l of 99% methanol was added to each plate well for 15min to fix the biofilm. After removing methanol, stain with 1% Crystal Violet (CV) solution for 30min, remove free stain, wash twice with deionized water, then elute biofilm with 200 μ Ι 33% acetic acid, transfer solution to clean 96-well plates to quantify the amount of biofilm formation. Quantification of biofilm formation Using a Lambda 35UV/VIS (USA) spectrophotometer at 590nm (OD) ₅₉₀ ) And (4) measuring.

The results of the biofilm inhibition test showed (a in fig. 5) that the color in the wells changed from light purple to black purple corresponding to the increase in the dilution concentration (25, 50, 125, 250 and 500 times), i.e., the increase in the biofilm formation amount. Based on the percentage of biofilm inhibition, the inhibition activity on biofilm formation was highest at 25-fold dilution of the cell-free supernatant (B in fig. 5), followed by 50, 125, 250 and 500-fold dilutions of the cell-free supernatant, respectively. Furthermore, there was no significant difference between the 250-fold and 500-fold dilutions of cell-free supernatant (P < 0.05).

And II, a pathogenic bacterium motility inhibition test.

To determine the inhibition of pathogenic bacteria motility by the cell-free fermentation broth produced by Bacillus velezensis JH22, a motility inhibition test was performed on semi-solid SM medium (3g/L beef extract, 5g/L peptone, agar of various concentrations and 20% glucose 25 ml/L). To determine the inhibitory effect of the cell-free fermentation broth on the motility of pathogenic bacteria dickinia daddii, SM media containing cell-

free supernatants

20, 40, 100, 200, and 400 times, respectively, and containing 0.3% agar were prepared. Plates without cell-free supernatant served as controls, and each treatment was repeated 3 times. After the plates had set, 2. mu.l of pathogenic bacterium Dietzia dadantii (10) was added to the center of each plate ⁸ CFU/mL), placing the plate in an incubator at 30 ℃ and culturing for 12 h. Then, the colony diameter for the motility of dickinia dadantii was measured, and the inhibition rate was calculated. Inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter × 100%.

The results of the cell-free fermentation liquid on the pathogenic bacteria motility inhibition effect measurement (figure 6) show that the cell-free fermentation liquid of different treatments has obvious inhibition effect (P is less than 0.05) on the motility of the pathogenic bacteria Ddaniella dicus (A in figure 6), the 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilution has 92.6 percent, 88.6 percent, 81.2 percent, 68.8 percent and 62.5 percent on the pathogenic bacteria motility inhibition effect respectively, but no obvious difference exists between the 20-fold and 40-fold dilution (B in figure 6).

Similarly, to determine the effect of cell-free fermentation broth on the suppression of colonization by the pathogenic bacterium dickinia dadditii, SM media containing 20, 40, 100, 200 and 400-fold cell-free supernatants, respectively, and containing 0.5% agar were prepared. Plates without cell-free supernatant served as controls and were repeated 3 times for each treatment. After the plates had set, 2. mu.l of pathogenic bacterium Dietzia dadantii (10) was added to the center of each plate ⁸ CFU/mL) of the bacterial liquid,the plate was placed in an incubator at 30 ℃ and incubated for 12 hours. The colony diameter for the motility of dickinia dadantii was measured, and the inhibition rate was calculated as above.

The results of the measurements of the colony motility inhibiting effect of the cell-free fermentation broth on the pathogenic bacteria show (FIG. 7) that the cell-free fermentation broths treated differently have significant inhibiting effect (P <0.05) on the colony motility of the pathogenic bacteria Dieckia dadditiva (A in FIG. 7), and the 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilutions have 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 bacterium of the dickinsonia dantii forms a biofilm after moving and clustering movement reaches the surface of a host under the stimulation of host secretion, and the generation of the biofilm promotes the interaction of the pathogenic bacterium and the host and then infects the host. The research provides that Bacillus velezensis JH22 can inhibit the mobility, the clustering movement and the biofilm formation of pathogenic bacteria, and provides another theoretical basis for the strain as a biocontrol bacterium.

Example 5

First, greenhouse experiment for biocontrol controlling stem and root rot of sweet potato.

In order to determine the control effect of the in-vivo biocontrol strain, a Bacillus velezensis JH22 strain is selected for a greenhouse disease control test. Selecting healthy sweet potato (Zhejiang potato No. 13, a susceptible variety) and intact potato, sterilizing with 75% alcohol for 5min, and washing with sterile water. Sterilizing the surface with 2% sodium hypochlorite solution for 5min, washing with sterile water for 3 times, and air drying in a clean bench. Sowing potato blocks in a vegetable cultivation plastic basket containing nutrient soil. The baskets were then moved to 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 the potato blocks (the base part of the seedling contains a small amount of potato block residues), and transplanting the seedlings into a new flowerpot containing nutrient soil. Using sterile needle to prick the base of each seedling stem, 10mL (10) of the prepared Bacillus belgii microbial inoculum ⁷ CFU/mL) was poured to the stem base of each sweet potato seedling, and 10mL of sterilized water was inoculated as a control. And respectively irrigating the prepared pathogenic bacterium dieldiella dadanensis microbial inoculum after 24 hours10mL(10 ⁷ CFU/mL) at the base of each respective seedling stem. Each treatment was 30 plants, each treatment was repeated 3 times, and the culture conditions were the same as above. After the disease occurs, observing the disease occurrence symptoms every day, and after 20 days, counting the disease incidence, the disease severity and the prevention and treatment effect of the disease. The disease grading standard is as follows (adopting a 0-4 grade grading scale):

0 is healthy and asymptomatic;

1, the overground part is healthy, and visible brown symptoms appear only around the base of the ground stem;

2, the stem base is dark brown, and yellow leaves or leaves on the upper part of the plant curl or droop;

the leaves of the plants on the ground are seriously yellowed or the plants are withered, so that the rotting of the stem base is serious;

whole plants wither or die.

Incidence (%) — number of diseased plants/total number of plants × 100%;

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

the preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index x 100%.

The greenhouse experiment result of biocontrol controlling the sweet potato stem root rot shows (figure 8) that the biocontrol microbial inoculum can delay the leaf symptoms and obviously reduce the incidence rate and the severity of the sweet potato stem rot. The control group inoculated with pathogenic bacteria showed harmful symptoms 57 days after inoculation, the stem base near the soil line showed dark brown symptoms, and the treated group inoculated with the inoculant showed no disease symptoms. After 11 days of inoculation, the treated group (the irrigating bacterial agent) part of the plants showed similar harmful symptoms, while the control group part of the plants showed severe disease symptoms. After 20 days, most of the control treated plants are yellow, the rotten symptoms of stems and roots are severe, and withered or dead individual plants appear (A in figure 8), while the inoculated biocontrol bacteria do not appear the withered symptoms, and only individual leaves are yellow (B in figure 8), thereby showing good biocontrol effect. The statistical results of disease control show (table 4), the biological control reduces the disease incidence by 37%, the disease index by 27%, and the control effect by 54.1%. Meanwhile, the biocontrol microbial inoculum is observed to remarkably promote the growth of plants.

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

And secondly, the growth promotion effect of the biocontrol strain on the growth of the sweet potatoes.

To evaluate the Plant Growth Promoting (PGP) ability of Bacillus velezensis JH22 inoculum, PGP test was performed under greenhouse conditions (same conditions as above). The cultivation method of the Zhejiang yam 13 seedling is the same as above. After 30 days of greenhouse cultivation, sweet potato seedlings with similar seedling length and size are selected, stolen from the stem base and cultivated in a pot containing nutrient soil. After planting, 10mL of Bacillus velezensis JH22 microbial inoculum (about 10) ⁷ CFU/mL). The preparation method of the microbial inoculum is the same as that of the microbial inoculum. And irrigating 10mL of the microbial inoculum once after 10-15 days. The same volume of sterile water was poured as a control. Each 30 sweet potato seedlings were treated as one treatment, and each treatment was repeated 3 times. After 45 days of cultivation, the growth promoting effect is counted. To determine the dry weight of the plant organs, they were placed in an oven and dried at 65 ℃ for 3 days. The growth promoting effect was evaluated by percent difference, i.e.,% difference ═ treatment-control)/control × 100%.

The results of the growth promoting effect test show that the biocontrol microbial inoculum remarkably promotes the increase of plant biomass (figure 9), and the stem length, the root length, the fresh weight, the dry weight, the fresh weight and the dry weight of the sweet potato are respectively increased by 30.3%, 28.8%, 21.1%, 49.7%, 54.7% and 65.0% after 45 days (table 5).

TABLE 5 growth promoting effect of biocontrol microbial inoculum on sweet potatoes

Aiming at the current situation that the stem and root rot of the sweet potato is difficult to prevent and treat, the invention screens out a Bacillus beiLeisi strain JH22 which can effectively prevent and treat the stem and root rot caused by pathogenic bacterium Dickinsonia dadantii, and the biocontrol agent can effectively inhibit the formation of a biological membrane of the Dickinsonia dadantii and the mobility and the clustering movement of thalli, and the biocontrol agent or biopesticide developed by utilizing the strain has good application prospect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

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

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

<130>

<160> 18

<170> PatentIn version 3.3

<210> 1

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<212> DNA

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agagtttgat cctggctcag 20

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aaggaggtga tccagccgca 20

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cagtcaggaa atgcgtacgt cctt 24

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caaggtaatg ctccaggcat tgct 24

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gacgtgggat ggctacaact 20

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attgtcgcct ttaacgatgg 20

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<212> DNA

<213> Artificial sequence

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<400> 7

acagagcttg gcgttgaagt 20

<210> 8

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<212> DNA

<213> Artificial sequence

<220>

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<400> 8

gcttcttggc tgaatgaagg 20

<210> 9

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<212> DNA

<213> Artificial sequence

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<400> 9

ttgtcgctca yaatgcaagc 20

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<212> DNA

<213> Artificial sequence

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ytcaagcatt tcrtctgtcg 20

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<212> DNA

<213> Artificial sequence

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gagcttgaag tkgttgaagg 20

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<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. The Bacillus belgii is characterized by being classified and named as Bacillus velezensis, the strain number is JH22, and the deposit number is as follows: CCTCC NO: m2022299.

2. Use of bacillus belgii according to claim 1 for antagonizing dieldia dadanii (Dickeya dadantii).

3. Use of the Bacillus belgii of claim 1 for the control of sweet potato stem root rot.

4. The use of Bacillus belgii according to claim 1 for sweet potato growth promotion.

5. The use according to claim 3 or 4, wherein a microbial agent comprising Bacillus belgii according to claim 1 is applied to the roots or stem bases of sweet potato seedlings.

6. A fungicide for controlling stem and root rot of sweet potato, comprising the Bacillus belgii of claim 1.

7. The microbial agent according to claim 6, wherein the concentration of Bacillus belgii is not less than 10 ⁷ CFU/mL。

8. A sweet potato biological control or sweet potato growth promotion method is characterized by comprising the following steps: selecting a cutting potato seedling having a length of three to four knots, and irrigating a microbial inoculum comprising the Bacillus belgii of claim 1 onto the roots of the sweet potato seedling.

9. The method for biocontrol of sweet potato or promoting growth of sweet potato as claimed in claim 8, wherein the concentration of Bacillus belgii in said microbial agent is not less than 10 ⁷ CFU/mL。

10. The sweet potato biocontrol or sweet potato growth promoting method according to claim 8, wherein the concentration of Bacillus belgii in said microbial agent is 10 ⁷ CFU/mL; the microbial inoculum is irrigated once again every 10 to 15 days; the irrigation amount is 5-15 mL per plant.