CN114933982B - Bacillus Velez and its application in preventing and controlling sweet potato stem and root rot - Google Patents

Abstract

The invention discloses a Bacillus velezensis and an application thereof in preventing and treating sweet potato stem and root rot. The Bacillus velezensis is classified and named as Bacillus velezensis, the strain number is JH22, and the deposit number is CCTCC NO: M 2022299. The Bacillus velezensis JH22 screened by the invention has a highly effective antagonistic effect on Dicteria davidianensis, can be used for preventing and treating sweet potato stem and root rot caused by Dicteria davidianensis, and the prevention effect in a greenhouse pot test reaches 54.1%. In the early stage of plant disease, it has an ideal prevention and treatment effect, and this characteristic indicates that the strain has great application potential as a biological preventive pesticide.

Description

Bacillus Velez and its application in preventing and controlling sweet potato stem and root rot

Technical Field

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

Background technique

Sweet potatoes are a strategically important staple crop that is grown worldwide. Asia is the second largest sweet potato-growing continent in the world, and China is the world's largest sweet potato producer, accounting for 56.6% of the world's total sweet potato production. Sweet potatoes are rich in carbohydrates, high-quality protein, dietary fiber, carotene, and trace elements such as potassium, zinc, calcium, and iron, which can provide important nutrients for the human body and provide energy for life activities.

Sweet potato stem and root rot is a bacterial disease caused by Dickeadadantii of the genus Dickeya, formerly known as Erwinia chrysanthemi. The disease seriously affects the yield and quality of sweet potatoes. The disease generally occurs in fields with a 10% to 20% incidence rate, while the incidence rate in severely affected fields is more than 50%, leading to a total crop failure in severe cases. The disease poses a huge threat to sweet potato production and food safety in sweet potato producing areas such as Zhejiang, Guangdong, Jiangsu, and Shandong provinces in my country. The pathogen has a wide host range. As of 2016, it has harmed ornamental plants in the Convolvulaceae, Asteraceae, and Orchidaceae, as well as vegetable plants in the Solanaceae, Leguminosae, Gramineae, and Cruciferae families, totaling more than 60 species of plants, including sweet potatoes, chrysanthemums, tobacco, peppers, tomatoes, potatoes, cabbages, eggplants, soybeans, morning glory, and dodder.

The pathogen mainly invades through the wounds of the host, producing pectinase to degrade the pectin in the plant cell wall, which causes the vascular bundle to become hollow and rot, causing the stems to turn dark brown, the plants to wilt, and have a rancid odor. Although the pathogen cannot survive in the soil for a long time, it can survive around plant debris, weeds, or the roots of other plants. In addition, the primary infection source may include diseased potatoes, diseased vines, and equipment contaminated during inter-irrigation farming operations.

Due to the wide host range and diversity of the pathogens of sweet potato stem and root rot, there is currently no effective prevention and control method for stem and root rot. Since the sweet potato cultivation area is still limited compared to crops, there is still a lack of research on diseases and disease-resistant breeding, and there are no disease-resistant varieties available in production. Disease control mainly relies on fungicides, such as 0.3% tetramycin, 72% agricultural streptomycin and 6% kasugamycin and other commonly used bacterial fungicides. The widespread use of fungicides not only kills beneficial microorganisms in the soil, but also causes the development of drug resistance in pathogens, and the use of large amounts of fungicides also poses potential hazards to ecology and human health. In recent years, a large number of studies at home and abroad have shown that the use of antagonistic microorganisms to develop biocontrol agents has become an inevitable trend of development, such as the use of Trichoderma fungi, Pseudomonas and Bacillus bacteria for biological control of plant diseases such as pepper blight, rapeseed sclerotinia, wheat scab, and rice blast. Sweet potato stem and root rot is a devastating disease. After infection by pathogens, the disease develops rapidly and can cause serious economic losses in a short period of time. However, there are relatively limited reports on the use of biological control methods to control sweet potato stem and root rot at home and abroad. In order to effectively prevent and control the serious occurrence of this disease, we carried out research on biological control of this disease. Based on field and indoor disease research, we developed a disease classification standard; screened biocontrol strains and studied their antagonistic mechanisms; and conducted disease control experiments, providing a reliable theoretical basis for the application of biocontrol strains. The development and application of biocontrol bacteria will provide a broad market and prospect for the prevention and control of sweet potato stem and root rot.

Summary of the invention

The invention provides a Bacillus Velezii that can be used for preventing and treating sweet potato stem and root rot. The screened strain has a highly effective antagonistic effect on Dictyopsis davidianensis and can be used for preventing and treating the sweet potato stem and root rot caused by Dictyopsis davidianensis, aiming to provide a new idea and new means for solving the sweet potato stem and root rot.

A type of Bacillus velezensis, classified and named Bacillus velezensis, with strain number JH22, was deposited in the China Center for Type Culture Collection in Wuhan on March 22, 2022, with the deposit number CCTCC NO: M2022299.

Biological and morphological characteristics of the strain:

The strain JH22 was cultured at a constant temperature (30°C) on a NA plate for 24 h. Its colonies were irregular circles, easily aggregated into lines, milky white, opaque, with a dry surface, and the inoculation ring was slightly sticky to the touch and had wrinkles on the edges.

Genetic characteristics of this strain:

The 16S rRNA sequence of strain JH22 is shown in SEQ ID NO: 13; the gyrA gene sequence is 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: 16; the groEL gene sequence is shown in SEQ ID NO: 17; and the polC gene sequence is shown in SEQ ID NO: 18.

The present invention also provides an application of the Bacillus Velezii in antagonizing pathogenic bacteria Dickeya dadantii, such as being used to prepare a bacterial agent for antagonizing pathogenic bacteria Dickeya dadantii ZJ97.

The invention also provides an application of the Bacillus Velezii in preventing and treating sweet potato stem and root rot.

The present invention also provides an application of the Bacillus Velezii in promoting the growth of sweet potatoes.

Optionally, the bacterial agent containing the Bacillus Velez subtilis is applied to the roots or stem bases of sweet potato seedlings.

The invention also provides a bacterial agent for preventing and treating sweet potato stem and root rot, comprising the Bacillus Velezii.

Optionally, the concentration of Bacillus velez 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 promotion method: selecting cutting potato seedlings with a length of three to four nodes, and pouring the bacterial agent containing the Velez bacillus onto the roots of the sweet potato seedlings.

Optionally, the concentration of Bacillus Velez subtilis in the bacterial agent is not less than 10 ⁷ CFU/mL.

Further optionally, the concentration of Bacillus Velez in the bacterial agent is about 10 ⁷ CFU/mL; the bacterial agent is irrigated once every 10 to 15 days; and the irrigating amount is 5 to 15 mL (preferably 10 mL) per plant.

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

(1) The Bacillus velezensis JH22 screened by the present invention has a highly effective antagonistic effect on Dictyopsis davidianensis and can be used to prevent and treat sweet potato stem and root rot caused by Dictyopsis davidianensis. Through conventional bacterial wound inoculation control tests and greenhouse potted plant tests, the control effect reached 54.1%. In the early stage of plant disease, it has an ideal control effect. This property shows that the strain has great application potential as a biological preventive microbial agent;

(2) The Bacillus velezensis JH22 screened by the present invention has a significant inhibitory effect on the growth of the pathogen Dictyosphaeria davidantii, with an inhibition rate of 75.8%. Similarly, its sterile cell supernatant has an inhibitory effect on the growth of pathogens of 68.8%;

(3) The Bacillus velezensis JH22 screened by the present invention has a protective effect of 54.1% against sweet potato pathogens under infection conditions;

(4) The expression level of the antagonistic compound of Bacillus velezensis JH22 screened by the present invention is high;

(5) The Bacillus velezensis JH22 screened by the present invention has a good growth-promoting effect on sweet potatoes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the in vitro inhibitory effect of five biocontrol bacteria on Dictyopsis davidantii (A: strain JH22, B: strain JH38, C: strain JH59, D: strain JH35, E: strain JH16);

Figure 2 is a morphological observation diagram of strain JH22 on NA plates;

FIG3 is a phylogenetic tree constructed by combining the sequences of gyrA gene, rpoB gene, purH gene, groEL gene and polC gene of Bacillus velez;

FIG4 is a MALDI-TOF-MS peak spectrum of lipopeptide compounds of strains JH22 and JH38 (A: JH22, B: JH38);

FIG5 is a graph showing the effect of strain JH22 on biofilm formation of pathogenic bacteria Dictyosphaeria davidantii (A: color change in wells at different dilution multiples, B: percentage inhibition of biofilm formation);

FIG6 is a graph showing the effect of strain JH22 on the motility of pathogenic bacteria Dictyosphaeria davidantii;

FIG7 is a graph showing the effect of strain JH22 on the swarming movement of pathogenic bacteria Dictyosphaeria davidantii;

FIG8 is a graph showing the control effect of strain JH22 on sweet potato stem and root rot in a greenhouse, FIGA is a control group, and FIGB is a treatment group;

FIG9 is a graph showing the growth-promoting effect of strain JH22 on sweet potato (the left side is the treatment group, and the right side is the control).

Detailed ways

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The soil environment is complex and diverse, and the effect of biological control of diseases usually depends on the local adaptability of the biocontrol agent to the soil. However, the poor competitiveness and soil adaptability of the introduced strains can only achieve limited success. For different pathogens in different regions, screening for efficient biocontrol strains is still a direction of concern for scientific researchers in various countries now and in the future. Therefore, the present invention screens out efficient biocontrol strains that are adapted to the local soil environment, and conducts relevant prevention and control research work according to local conditions, which is an important task for biological control of the disease.

Screening for antagonistic strains of sweet potato stem and root rot biological control can effectively control sweet potato stem and root rot, reduce the cost of disease control, and increase sweet potato yields. On the other hand, it can promote environmental protection, biosafety, and industrial production of products. Bacillus is an important member of bacterial biological control. It can be used as a biological control agent to replace chemical pesticides in some agricultural production to solve disease problems in plant production practices. Bacillus is also a commonly used plant growth-promoting rhizobacteria (PGPR), which can produce compounds that promote plant growth, provide biological nitrogen fixation, and trigger metabolic activities of plant roots. Research on Bacillus has good prospects for sustainable application.

Example 1

1. Strain isolation and screening.

The test soil samples were collected in December 2020 from a continuously cropped sweet potato field with severe disease in Wucheng District, Jinhua City, Zhejiang Province (28°48′14″N, 119°20′56″E). Soil samples near the roots of diseased plants were randomly collected in sterile bags, sealed and stored in a refrigerator at 4°C.

NA plate screening of biocontrol bacteria in soil: Take 1g of soil sample in sterile water, shake for 3min to prepare soil suspension, and dilute to a concentration of ^10-4 to ^10-6 . Spread the prepared soil suspension evenly on the NA plate with a sterile coating rod and grow at 30℃ for 48h. Select different single colonies according to the shape, color, size, regularity, concavity and other characteristics of the colony. Purify each colony 3 times, prepare bacterial solution and add 30% glycerol to store in a -40℃ freezer. A total of 301 strains were screened from 15 soil samples.

2. Screening of biocontrol strains.

The antagonistic activity of the isolated strains was evaluated by agar diffusion method. All the isolated strains were inoculated in liquid NA medium and cultured in a HZQ-F100 shaker at 30°C and 200rpm for 24h to obtain a bacterial solution (adjusted to about 10 ⁷ CFU/mL). Similarly, the pathogen Dictyosporidae ZJ97 was also inoculated in liquid NA medium and cultured under the above conditions. Take 400μl of Dictyosporidae bacterial solution (10 ⁸ CFU/mL) and spread it on the surface of a NA plate (9cm in diameter). Three equidistant Oxford cups (6mm in diameter) were inserted on its surface, and 25μl of the fermentation liquid of the strain to be screened was added to the Oxford cup wells. Each strain was repeated 3 times. After the culture dish was placed in a 30°C incubator and cultured for 24h, the diameter of the inhibition zone was observed and measured.

According to the diameter of the inhibition zone (diameter greater than 13.0 mm), a total of 5 highly active candidate strains were screened (Table 1), named JH16, JH22, JH35, JH38 and JH59, which all significantly inhibited the growth of pathogens (P<0.05), with inhibition effects of 59.6%, 75.8%, 56.5%, 69.8% and 63.2%, respectively.

To determine the activity of secondary metabolites of the five candidate strains, the bacterial suspensions were centrifuged at 4 °C and 6000 rpm for 10 min to obtain cell-free supernatant (CFS). The CFS was further filtered through a 0.22-μm filter. Similarly, the pathogenic bacterium Dictyosporidae ZJ97 was inoculated in liquid NA medium, three equidistant Oxford cups were inserted, and 25 μl of cell-free supernatant was added to the wells of the Oxford cups, as described above. After the culture dishes were placed in an incubator at 30 °C for 24 h, the diameter of the inhibition zone was observed and measured.

The results of the activity assay of secondary metabolites of the five candidate strains showed (as shown in Figure 1) that they all significantly inhibited the growth of pathogens (P<0.05), with the inhibitory effects of 55.7%, 68.8%, 52.3%, 62.9%, and 56.8%, respectively (Table 1). Since strains JH22 and JH38 had higher inhibitory activity, they were used for further research.

Table 1 The inhibitory effect of biocontrol strains on pathogenic bacteria Dictyopsis davidianensis

a: bacterial culture; b: cell-free supernatant

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

Example 2 Identification of strains.

Two strains with high activity were identified.

Morphological observation is shown in Figure 2. The strain JH22 was cultured on a NA plate at a constant temperature (30°C) for 24 hours, and its colonies were irregular circles. The colonies tended to accumulate into lines, were milky white, opaque, and had a dry surface. The inoculation ring was slightly sticky to the touch and had wrinkles on the edges.

To identify the two strains JH22 and JH38 molecularly, PCR amplification of 16S and five housekeeping genes was performed on them, respectively. The PCR amplification process is shown in Table 2. The PCR amplification reaction system was 50 μL: ddH ₂ O 17.5 μL, HLingene PCR Master Mix 25 μL, 16S-27f 2.5 μL, 16S-1492r 2.5 μL, and DNA template 2.5 μL. The PCR reaction program was 95°C for 5 min pre-denaturation; 95°C for 30 s denaturation; 56°C for 30 s annealing; 72°C for 60 s extension; 35 cycles; and 4°C storage. The PCR amplification products were bidirectionally sequenced by Beijing Qingke Biotechnology Co., Ltd. (Hangzhou Branch), and the 16S rRNA sequence of strain JH22 is shown in SEQ ID NO: 13. The obtained 16S sequence was compared with the homology sequence in GenBank by the Blast search engine to determine the corresponding genus.

To distinguish different closely related species within the genus, the sequences of five housekeeping genes were used, among which the gyrA gene sequence of strain JH22 is 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: 16; the groEL gene sequence is shown in SEQ ID NO: 17; and the polC gene sequence is shown in SEQ ID NO: 18. The gyrA gene, rpoB gene, purH gene, groEL gene and polC gene of closely related species were downloaded from GenBank (Table 2) for phylogenetic analysis. The downloaded Bacillus cereus ATCC14579 was used as an outgroup.

In order to construct the phylogenetic tree, the obtained sequences were edited with Clustalx1.83, the sequences of each gene were arranged with MAFFT 7.273 software, the ambiguous regions were removed with Gblocks 0.91b, the best GTR+I+G nucleotide substitution model was obtained with jModel Test 2.1.7, and finally the phylogenetic tree was constructed with RaxmlGUI v.1.5 software and the maximum likelihood (ML) method.

As shown in Figure 3, the two test isolates JH22 and JH38 clustered with Bacillus velezensis BD568, BD569, B23190 and B23189 to form a clear branch with a 100% bootstrap support value. Therefore, both strains were identified as Bacillus velezensis. Among them, JH22 was deposited in the China Center for Type Culture Collection in Wuhan on March 22, 2022, with the deposit number CCTCC NO: M 2022299.

Table 2 Primers for PCR amplification

Example 3 Detection of bacterial strain lipopeptide compounds.

Lipopeptides of candidate strains were determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bacillus velezensis JH22 and JH38 were inoculated on NA plates and cultured at 30°C for 24 h. Single colonies were picked and dissolved in centrifuge tubes (2.0 mL) containing matrix solution. The matrix solution contained 10 mg/mL cyano-4-hydroxycinnamic acid (dissolved in 70% acetonitrile) and 0.1% trifluoroacetic acid (TFA) in 70% acetonitrile. The solution was mixed and centrifuged at 5000 r/min for 2 min. Then 1 μL of the sample was aspirated and dropped onto a MALDI-TOF MTP 384 target plate (Germany) and the data were recorded using an ultrafleXtreme ^TM MALDI-TOF mass spectrometer (Germany) equipped with an intelligent beam laser. The measurements were performed in reflectron mode and with an ion source accelerating voltage of 20 kV. The mass spectral data were stored in the low mass range between 0.1 and 2 kD. Based on the mass spectrometry peaks of the lipopeptide series reported in the literature and determined, the lipopeptide compounds they produced were analyzed (Table 3).

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

Although strains JH22 and JH38 both produced three lipopeptide compounds, the intensity of the three lipopeptide compounds produced by strain JH22 was significantly higher than that of JH38 (Figure 4), indicating that strain JH22 produced more lipopeptide compounds than strain JH38, which was consistent with the above in vitro bioactivity test results. Therefore, strain JH22 was selected as the most promising biocontrol strain and was further used to study the inhibition of biofilm formation and motility of pathogens, as well as the biocontrol effect and growth promotion effect.

Table 3 Lipopeptide analysis results 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

1. Inhibitory effect on biofilm formation.

Preparation of biocontrol bacteria seed solution: Pick a single colony of Bacillus velezensis JH22 and inoculate it into 10 mL of liquid NA (no agar) liquid culture medium, and culture it at 30°C and 200 rpm with shaking for 24 hours until the OD ₆₀₀ of the culture solution is 0.6-0.8, which is used as the liquid fermentation seed solution of the biocontrol agent.

Liquid fermentation: 500 μL of biocontrol bacteria seed solution was inoculated into 500 mL of liquid NA (no agar) medium, and cultured at 30°C and 200 rpm for 60 hours to obtain biocontrol bacteria liquid fermentation solution. The concentration of Bacillus Velezii in the fermentation solution was adjusted to about 10 ⁷ CFU/mL to obtain the biocontrol agent.

Preparation of cell-free supernatant: The biocontrol bacteria liquid fermentation broth was centrifuged at 6000 rpm for 10 min, and then the residual bacteria were filtered using a 0.22 μm bacterial filter to obtain Bacillus velezensis JH22 cell-free supernatant.

In order to determine the effect of cell-free supernatant of biocontrol bacteria on biofilm formation of pathogens, a biofilm formation inhibition test was performed in a 96-well plate. First, 40 μl of cell-free supernatant diluted 5, 10, 25, 50 and 100 times was added to each well, followed by 20 μl of pathogen Dictyostomia davidii (10 ⁸ CFU/mL) and 140 μl of liquid NA (no agar) medium. Finally, the volume in each well reached 200 μl, and the cell-free supernatant was finally diluted to 25, 50, 125, 250 and 500 times, respectively. The addition of 40 μl of sterile saline was used as a control, and each treatment was repeated 3 times. After the 96-well plate was placed at 30°C for 24 hours, the culture medium was discarded, washed three times with sterile water, non-adherent cells were removed, air-dried in a clean bench, and then 100 μl of 99% methanol was added to each well for 15 minutes to fix the biofilm. After removing the methanol, the plate was stained with 1% crystal violet (CV) solution for 30 minutes, free staining was removed, and the plate was washed twice with deionized water. The biofilm was then eluted with 200 μl of 33% acetic acid, and the solution was transferred to a clean 96-well plate to quantify the amount of biofilm formation. The quantification of biofilm formation was measured at 590 nm (OD ₅₉₀ ) using a Lambda 35UV/VIS (USA) spectrophotometer.

The results of the biofilm inhibition test showed (Figure 5 A) that the color in the wells changed from light purple to dark purple corresponding to the increase in dilution concentration (25, 50, 125, 250 and 500 times), that is, the increase in the amount of biofilm formation. According to the percentage of biofilm inhibition, the 25-fold dilution of the cell-free supernatant had the highest inhibitory activity on biofilm formation (Figure 5 B), followed by 50, 125, 250 and 500-fold dilutions of the cell-free supernatant. In addition, there was no significant difference between the 250-fold and 500-fold dilutions of the cell-free supernatant (P<0.05).

2. Pathogen motility inhibition test.

In order to determine the inhibitory effect of cell-free fermentation broth produced by Bacillus velezensis JH22 on the motility of pathogenic bacteria, a motility inhibition test was carried out in semi-solid SM medium (3g/L beef extract, 5g/L peptone, different concentrations of agar and 20% glucose 25ml/L). In order to determine the inhibitory effect of cell-free fermentation broth on the motility of pathogenic bacteria, SM medium containing 20, 40, 100, 200 and 400 times of cell-free supernatant and 0.3% agar was prepared. Plates without cell-free supernatant were used as controls, and each treatment was repeated 3 times. After the plates solidified, 2μl of pathogenic bacteria Dadantidix (10 ⁸ CFU/mL) was added to the center of each plate, and the plates were placed in a 30℃ incubator and cultured for 12h. Then, the colony diameter of Dadantidix motility was measured and the inhibition rate was calculated. Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

The results of the determination of the inhibitory effect of cell-free fermentation broth on pathogen motility showed (Figure 6) that the cell-free fermentation broths treated with different methods had a significant inhibitory effect on the motility of the pathogen Dictyoshi I. davidii (P<0.05) (Figure 6A). The inhibitory effects of 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilutions on pathogen motility were 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 (Figure 6B).

Similarly, in order to determine the inhibitory effect of cell-free fermentation broth on the swarming movement of pathogenic bacteria Dictyopsis davidiansis, SM medium containing 20, 40, 100, 200 and 400 times of cell-free supernatant and 0.5% agar was prepared. Plates without cell-free supernatant were used as controls, and each treatment was repeated 3 times. After the plates solidified, 2 μl of pathogenic bacteria Dictyopsis davidiansis (10 ⁸ CFU/mL) was added to the center of each plate, and the plates were placed in a 30°C incubator and cultured for 12 hours. The colony diameter of Dictyopsis davidiansis was measured, and the inhibition rate was calculated as above.

The results of the determination of the inhibitory effect of cell-free fermentation broth on the swarming movement of pathogens showed (Figure 7) that the cell-free fermentation broths treated with different treatments had a significant inhibitory effect on the swarming movement of the pathogen Dictyosphaeria davidianta (P<0.05) (Figure 7A). The inhibitory effects of 20-fold, 40-fold, 100-fold, 200-fold and 400-fold dilutions on the movement of pathogens were 93.4%, 90.4%, 81.1%, 73.2% and 64.0%, respectively, but there was no significant difference between the 20-fold and 40-fold dilutions (Figure 7B).

The pathogenic bacterium Dictyosphaeria davidianta, stimulated by host secretions, forms a biofilm after reaching the host surface through swimming and swarming. The formation of the biofilm promotes the interaction between the pathogen and the host, and then infects the host. This study provides that Bacillus velezensis JH22 can inhibit the motility, swarming and biofilm formation of pathogens, providing another theoretical basis for this strain as a biocontrol bacterium.

Example 5

1. Greenhouse experiment on biological control of sweet potato stem and root rot.

In order to determine the control effect of the biocontrol strain in vivo, the Bacillus velezensis JH22 strain was selected for greenhouse disease control experiments. Healthy sweet potato (Zheshu No. 13, a susceptible variety) tubers were selected and sterilized in 75% alcohol for 5 minutes, and then rinsed with sterile water. The tubers were then surface sterilized with 2% sodium hypochlorite solution for 5 minutes, rinsed with sterile water 3 times, and air-dried in a clean bench. The tubers were sown in a vegetable cultivation plastic basket containing nutrient soil. The basket was then moved to a glass greenhouse with a temperature of 25-28°C and a relative humidity of 80-90%. After 30 days (4-5 leaf stage), seedlings with consistent height were selected, and each seedling was cut from the tuber (the base of the seedling contained a small amount of tuber residue) and transplanted into a new pot containing nutrient soil. Use a sterile needle to pierce the base of each seedling stem, and pour 10mL (10 ⁷ CFU/mL) of the prepared Bacillus Velez inoculum into the base of each sweet potato seedling stem, and inoculate 10mL of sterilized water as a control. And after 24 hours, pour 10mL (10 ⁷ CFU/mL) of the prepared pathogenic bacteria Dadanti Dixon inoculum into the base of each corresponding seedling stem. Each treatment consists of 30 plants, and each treatment is repeated 3 times. The culture conditions are the same as above. After the disease occurs, observe the symptoms of the disease every day. After 20 days, the incidence, severity and control effect of the disease are statistically analyzed. The disease grading standard is as follows (using a 0-4 rating scale):

0 = healthy plant, no symptoms;

1 = The aboveground plant is healthy, with only visible brown symptoms around the base of the stem above ground;

2 = Dark brown symptoms are obvious at the base of the stem, with yellowing leaves or curled or drooping leaves on the upper part of the plant;

3 = The leaves of the above-ground plants are severely yellow or the plants wilt, and the base of the stems are severely rotten;

4 = The entire plant wilts or dies.

Incidence rate (%) = number of diseased plants/total number of plants × 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 × 100%.

The results of the greenhouse experiment on the biocontrol of sweet potato stem and root rot showed (Figure 8) that the biocontrol agent can delay leaf symptoms and significantly reduce the incidence and severity of sweet potato stem diseases. The control group inoculated with pathogens showed damage symptoms 57 days after inoculation, and dark brown symptoms appeared at the base of the stem close to the soil line, while the plants in the treatment group irrigated with the agent showed no disease symptoms. 11 days after inoculation, some plants in the treatment group (irrigated with the agent) showed similar damage symptoms, while some plants in the control group showed serious disease symptoms. After 20 days, most of the leaves of the control plants turned yellow, and the stem and root rot symptoms were serious. Some plants withered or died (Figure 8 A), while the inoculated biocontrol bacteria did not show wilting symptoms, and only some leaves turned yellow (Figure 8 B), showing a good biocontrol effect. The statistical results of disease control showed (Table 4) that biological control reduced the incidence of diseases by 37%, reduced the disease index by 27%, and the control effect was 54.1%. At the same time, it was also observed that the biocontrol agent significantly promoted the growth of plants.

Table 4 Effect of biocontrol agents on greenhouse control of sweet potato stem and root rot

2. The growth-promoting effect of biocontrol strains on sweet potato growth.

In order to evaluate the plant growth promoting (PGP) ability of Bacillus velezensis JH22 inoculant, a PGP test was conducted under greenhouse conditions (conditions as above). The cultivation method of Zhejiang Shu No. 13 seedlings was the same as above. After 30 days of greenhouse cultivation, sweet potato seedlings of similar seedling length and size were selected, stolen from the base of the stem, and cultivated in pots containing nutrient soil. After planting, 10 mL of Bacillus velezensis JH22 inoculant (about 10 ⁷ CFU/mL) was poured. The preparation method of the inoculant was the same as above. After 10 to 15 days, 10 mL of the inoculant was poured again. The same volume of sterilized water was poured as a control. Every 30 sweet potato seedlings were used as a treatment, and each treatment was repeated 3 times. After 45 days of cultivation, the growth-promoting effect was counted. In order to determine the dry weight of plant organs, they were placed in an oven and dried at 65°C for 3 days. The growth-promoting effect was evaluated by percentage difference, that is, percentage difference % = (treatment-control)/control×100%.

The results of the growth-promoting effect test showed that the biocontrol agent significantly promoted the increase of plant biomass (Figure 9). After 45 days, the stem length, root length, seedling fresh weight, seedling dry weight, root fresh weight and root dry weight of sweet potato increased by 30.3%, 28.8%, 21.1%, 49.7%, 54.7% and 65.0%, respectively (Table 5).

Table 5 Effect of biocontrol agents on the growth promotion of sweet potato

In view of the current situation that sweet potato stem and root rot is difficult to prevent and treat, the present invention screens out a Velezii bacillus strain JH22 which can effectively prevent and control the stem and root rot caused by the pathogenic bacterium Dixidiella davidanti, and the biocontrol agent thereof can effectively inhibit the formation of biofilm of Dixidiella davidanti and the motility and cluster movement of bacteria, and the biocontrol agent or biological pesticide developed by using the strain has a good application prospect.

The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

SEQUENCE LISTING

<110> Jinhua Academy of Agricultural Sciences (Zhejiang Agricultural Machinery Research Institute)

<120> A kind of Bacillus Velezii and its application in preventing and controlling 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

cttgccggcgcgcgtagctgt 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 (8)

1. A Bacillus velezensis, characterized in that it is classified and named Bacillus velezensis , the strain number is JH22, and the deposit number is: CCTCC NO: M 2022299. 2. Use of the Bacillus Velezii as claimed in claim 1 in antagonizing Dickeya dadantii . 3. Use of Bacillus Velezii as claimed in claim 1 in preventing and treating sweet potato stem and root rot. 4. Use of Bacillus Velez in promoting sweet potato growth as claimed in claim 1. 5. The use according to claim 3 or 4, characterized in that the bacterial agent comprising the Bacillus Velez subtilis according to claim 1 is applied to the roots or stem bases of sweet potato seedlings. 6. A bacterial agent for preventing and treating sweet potato stem and root rot, characterized in that it comprises the Bacillus Velezii as described in claim 1. The bacterial agent according to claim 6, characterized in that the concentration of the Bacillus Velez subtilis is not less than 10 ⁷ CFU/mL. 8. A method for biological control of sweet potato or promoting growth of sweet potato, characterized in that it comprises: selecting cutting potato seedlings with a length of three to four nodes, and pouring the bacterial agent containing the Bacillus Velez subtilis according to claim 1 onto the roots of the sweet potato seedlings; the concentration of the Bacillus Velez subtilis in the bacterial agent is not less than 10 ⁷ CFU/mL; pouring the bacterial agent again every 10 to 15 days; and pouring at a rate of 5 to 15 mL per plant.