TWI705137B - Bacterial strain for preventing and controlling plant diseases, microbial composition, microbial fertilizer, and method for using the same - Google Patents

Abstract

A bacterial strain ( Bacillus velezensis,HL_B03) for preventing and controlling plant diseases, a microbial composition, and a method for using the same are provided. Wherein, the bacterial strain can antagonize bacterial Ralstonia solanacearum, the microbial composition includes the bacterial strain and a rapeseed meal. The method for using the microbial composition includes applying the microbial composition to soil for crop growing, or applying on surface of crop, thereby increase the nutrition of soil and facilitate crop against bacterial diseases.

Description

Strains, microbial compositions and methods of use for plant disease control

The present invention relates to a strain (Bacillus HL_B03), a microbial composition and a method of use thereof, and particularly relates to a strain, a microbial composition, and method of use that contribute to the prevention and control of plant diseases.

Plant diseases of crops cause yellowing, dwarfing, leaf deformation, fruit deformation and withering of crops. Among them, soil-borne bacterial wilt is particularly serious. Ralstonia solanacearum invades plants through plant roots and multiplies in the xylem, eventually causing wood blockage and causing the plants to wither and die. In addition, the bacterial wilt can survive in the soil for a long time, so once it occurs, it is difficult to eradicate the bacterial wilt.

Traditionally, chemical fertilizers are often used for the prevention and control of plant diseases of crops, but this often causes soil acidification and solidification, and drastically reduces the number of soil microorganisms, resulting in the destruction of soil texture, which affects its ability to store water and maintain fertilizer. Thus hindering the growth of crops. Further, the traditional soil fumigant is also a chemical reagent (for example, methyl bromide), which is widely used in the sterilization and sterilization of soil. Although its control effect is excellent, it has the risk of destroying the ozone layer and causing the imbalance of soil microorganisms. In summary, the development of a microbial composition that can improve soil nutrient status and help crops antibacterially is an important topic in the field.

In order to solve the above-mentioned problems, the purpose of the present invention is to provide a strain (Bacillus HL_B03), microbial composition and method of use for the prevention and control of plant diseases, so as to improve the problems of conventional techniques and thereby increase the yield and quality of crops.

According to one objective of the present invention, a Bacillus strain (HL_B03) is proposed, which is deposited at the Food Industry Development Institute of a consortium legal person, with the deposit label BCRC910810, wherein the strain can antagonize bacterial wilt.

Preferably, the strain is further obtained by a screening method. The screening method includes (1) providing soil containing Bacillus to the culture medium; (2) culturing at 30° C. for 24 to 48 hours, adding 10 ⁸ cfu/ml Ralstonia solanacearum The pathogens are in the culture medium; (3) the initial strains with the radius of the antagonistic circle close to or greater than 1cm are selected; (4) the initial strains are cultivated in a medium containing rapeseed meal (rapeseed meal) and cultured at 30°C for 24~ 48 hours; and (5) Select strains that can produce utilization circles.

According to another object of the present invention, a microbial composition for the prevention and control of plant diseases is provided, which includes a Bacillus strain (HL_B03) and rapeseed meal; wherein the proportion of the strain in the microbial composition is 2 to 3%. The proportion of seed meal in the microbial composition is 97-98%.

Preferably, in the microbial composition for plant disease control, the rapeseed meal includes at least one of mustard powder, rapeseed meal, and canola meal.

Preferably, in the microbial composition for plant disease control, the plant disease includes bacterial wilt or loofah wilt.

Preferably, the microbial composition further includes a base fertilizer, wherein the weight ratio of the microbial composition to the base fertilizer is 3:5-9:10.

According to another object of the present invention, a method for using a microbial composition is proposed, which includes (1) applying the above-mentioned microbial composition to the soil where crops are scheduled to be grown; or (2) applying the above-mentioned microbial composition to the surface of the crop.

Preferably, in the method of using the microbial composition, the crops include Solanaceae or Cucurbitaceae crops.

The strain (Bacillus HL_B03), microbial composition and method of use for plant disease control of the present invention have the following advantages:

(1) The strain of the present invention has the ability to antagonize bacterial wilt and can effectively control bacterial wilt caused by the bacteria. In addition, the strain is a kind of phosphate solubilizing bacteria that can decompose insoluble substances in the soil and convert them into phosphorus, iron, calcium, etc., to provide nutrients for crops and help them absorb it. Accordingly, the strain can be added to various fertilizers to enhance the efficacy of fertilizers, and further enhance the resistance of crops to plant diseases, especially the control of soil-borne pathogens.

(2) Rapeseed meal is a by-product of rapeseed oil extraction. It has antibacterial ability and can also be used as organic fertilizer. In the present invention, rapeseed meal can be used as a soil fumigant to replace chemical methyl bromide. Because it is a natural organic substance, it can avoid the shortcomings caused by chemical reagents, and can achieve the effect of soil microbial phase balance.

(3) In the microbial composition of the present invention, rapeseed meal is used as a biological fumigant, combined with the strain (Bacillus HL_B03), the combination of the two can synergistically control bacterial wilt, compared to individual use cases , Can provide better disease resistance.

In order to make the above objectives, technical features, and benefits after actual implementation easier for those with ordinary knowledge in the field to understand, the following examples will be used to illustrate in more detail with drawings.

The Bacillus strain (HL_B03) of the present invention is deposited at the Food Industry Development Research Institute, and the deposit number is BCRC910810. The strain can antagonize bacterial wilt. Further, the bacterial species identification was carried out by the Food Industry Development Institute of a consortium, and it was confirmed that the strain was Bacillus velezensis ; its 16s rDNA sequence was shown in SEQ ID NO:1 in the sequence table, and its gyrB gene sequence SEQ ID NO: 2 shown in the sequence listing.

The Bacillus strain of the present invention has the ability to antagonize bacterial wilt, and is obtained through a screening method. Please refer to Figure 1, which is a flowchart of the method for screening Bacillus strains of the present invention. The screening method may include: (S1) providing soil containing Bacillus to the culture medium; (S2) after culturing at 30°C for 24 to 48 hours, adding 10 ⁸ cfu/ml Ralstonia solanacearum to the culture medium; (S3) from The culture medium selects the initial strains whose antagonistic circle radius is close to or greater than 1cm; (S4) cultivates the initial strains in a medium containing rapeseed meal at 30°C for 24 to 48 hours; and (S5) selects from the aforementioned medium Strains capable of growing and producing utilization circles.

In an embodiment of the screening method of the present invention, soil samples from 807 places in Yilan and Hualien areas were collected as soil samples containing Bacillus. 1 g of soil was taken from each soil sample, and after mixing, 9 ml of soil was added. Treat sterile water in a 60°C water bath for 30 minutes. Here, the diluted plate method is used to smear the treated soil on a nutrient medium (Nutrient Agar, NA) and place it in an incubator at 30°C for culture. After culturing for 24 hours, spray 10 ⁸ cfu/ml of Ralstonia solanacearum on the medium evenly using a glass spray bottle, and then incubate for another 24 hours in an incubator at 30°C. Next, referring to Figure 2, when waiting for a complete antagonistic circle to appear, select strains with an antagonistic circle radius close to or greater than 1 cm. Among them, the source of bacterial wilt pathogen was isolated from tomato bacterial wilt diseased plants in Yilan, and its number was LE5. Then, the selected strains are used for subsequent plant disease control tests. The strains to be tested are cultivated on a nutrient medium containing 2% rapeseed meal. After 24 hours of cultivation, strains that can grow and produce utilization circles are selected.

In the verification test of the antagonistic effect of the strain of the present invention (Bacillus HL_B03), 10g of rapeseed meal was added to 100ml of sterile water to prepare a water extract of rapeseed meal, at 30°C, on a shaker , Shake at 250rpm for 24 hours for extraction. Fusarium oxysporum f. sp. Luffae (Luffarium oxysporum f. sp. Luffae ) was cultured to produce spores, and then washed with 1 mL of 0.01% tween20 to wash off the spores and prepared into an aqueous solution of Fusarium oxysporum f. sp. Luffae . Centrifuge 1 mL 10 ⁸ cfu/ml of the strain of the present invention at 4,000-6,000 rpm for 3 minutes, and collect the supernatant. Dilute with 0.01% tween20, dilute the aforementioned rapeseed meal water extract 80 times, dilute the Fusarium oxysporum aqueous solution to a concentration of 10 ⁶ spore/ml, and dilute the supernatant 500 times to prepare accordingly Four groups of solutions, the control group contains only Fusarium oxysporum, the rapeseed meal group contains Fusarium oxysporum and rapeseed meal, the HL_B03 group contains Fusarium oxysporum and the supernatant of the strain of the present invention, rapeseed The meal+HL_B03 group contains Fusarium oxysporum, rapeseed meal and the supernatant of the strain of the present invention. Drop the solutions of the four groups on a clean grooved glass slide, and cover with a lid. It was placed in a nutrient medium that maintains humidity, and the germination rate was investigated after 24 hours at 25°C. Each treatment is 6 repetitions, and the test is repeated 3 times.

The calculation method of spore germination rate is: spore germination rate (%) = the number of spores whose germination tube exceeds half the length of the spore/total number of spores x 100%. Please refer to Figure 3, which is the result of the experiment of inhibiting the spore germination of Fusarium oxysporum by the strain of the present invention (Bacillus HL_B03) against Luffa wilt. As shown in Figure 3, the rapeseed meal and the strain (Bacillus HL_B03) individually have the effect of inhibiting the germination of the spores of Fusarium oxysporum of Luffa wilt, and the combination of rapeseed meal and the strain (Bacillus HL_B03) further has Collaboration adds success.

The microbial composition of the present invention may include a strain (Bacillus HL_B03) and rapeseed meal. Wherein, the proportion of the strain in the microbial composition can be 2 to 3%, and the proportion of rapeseed meal in the microbial composition can be 97 to 98%. Rapeseed meal may include at least one of mustard powder, rapeseed meal, and canola meal, but is not limited thereto. The plant diseases that can be controlled include bacterial wilt or loofah wilt, but not limited to this. In addition, the microbial composition of the present invention may further include a basal fertilizer, wherein the weight ratio of the microbial composition to the basal fertilizer may be 3:5-9:10. Preferably, the weight ratio of the microbial composition to the base fertilizer can be 13:20-17:20.

In one embodiment, the source of rapeseed meal includes mustard meal, rapeseed meal, and canola meal. The aqueous extract of rapeseed meal is used to prepare the aqueous extract of rapeseed meal of mustard powder, rapeseed meal and canola meal with a concentration of 0.1 g/mL. A nutrient medium containing 2% of the three rapeseed meal mentioned above is individually configured. In addition, in the control group, sterile water was used to replace the water extract of rapeseed meal, a single colony of Bacillus (HL_B03) was taken to each medium, and then 10 ⁸ cfu/ml bacterial wilt bacteria were evenly sprayed with a glass spray bottle In each medium, incubate at 30°C for 24 hours, measure and record the diameter of the antagonistic circle. The results are shown in Figure 4. It can be seen from Figure 4 that the three kinds of rapeseed meal mentioned above all have antagonistic effects on bacterial wilt disease, among which rapeseed meal has the greatest antagonistic effect.

In one embodiment, the aforementioned water extract of rapeseed meal is diluted 500 times, 600 times, 800 times, and 1000 times to obtain 500 times, 600 times, 800 times, and 1000 times dilutions. Carry out the plate antagonism test of Ralstonia solanacearum. Among them, the experimental conditions are the same as the aforementioned experimental methods of rapeseed meal from different sources. Please refer to Figure 5, which shows that all three dilutions except the 1000-fold dilution have antagonistic effects on bacterial wilt, and the lowest dilution with antagonistic effect is 800-fold.

In the method of using the microbial composition of the present invention, it may include (1) applying the microbial composition to the soil where the crop is intended to be grown; or (2) may include applying the microbial composition to the surface of the crop; or may include the aforementioned A combination of (1) and (2). In addition, the crops may include Solanaceae or Cucurbitaceae crops, but not limited to this. For example, crops may include potatoes, eggplants, tomatoes, cucumbers, pumpkins, loofah, or watermelon.

In an embodiment of the method of using the microbial composition of the present invention, the morbidity test of Ralstonia solanacearum was conducted in a greenhouse. In the control group, two-week-old tomatoes were transplanted to culture soil, one plant per hole, and the bacterial wilt pathogen was watered at the same time. The rapeseed meal treatment group consisted of mixing rapeseed meal with a ratio of 1:800 in the cultivation soil before planting, and transplanting two-week-old tomatoes to the cultivation soil mixed with rapeseed meal one week later, planting one plant per hole, and watering the bacterial wilt at the same time ; This group is marked as rapeseed meal in Figure 6. The strain (Bacillus HL_B03) treatment group was transplanted two-week-old tomatoes to the culture soil for planting, one plant per hole, and the strain (Bacillus HL_B03) was watered at the same time, every 5 days, continuous watering 3-5 times; another for colonization Water Ralstonia solanacearum at time; this group is labeled HL_B03 in Figure 6. The treatment group of the combination of rapeseed meal and strain (Bacillus HL_B03) is to mix rapeseed meal with a ratio of 1:800 in the cultivation soil before planting, and transplant two-week-old tomatoes to the cultivation soil mixed with rapeseed meal one week later. One plant per hole, and water the strain (Bacillus HL_B03) at the same time, 3-5 consecutive watering every 5 days; watering the bacterial wilt fungus during the planting; this group is marked as rapeseed meal+HL_B03 in the 6th figure . Wherein the amount of bacterial wilt and watering concentrations as are 0.01L and 10 ⁶ cfu / mL. The watering amount and concentration of the aforementioned Bacillus HL_B03 are the same, both 0.01L and 10 ⁷ cfu/mL. And the morbidity rate is calculated as: morbidity rate (%) = number of diseased plants / total number of plants x 100%.

The results are shown in Figure 6, which shows that rapeseed meal and the strain (Bacillus HL_B03) individually have control effects on tomato bacterial wilt, and the combination of rapeseed meal and strain (Bacillus HL_B03) further has a synergistic effect. Successful.

In another embodiment of the method of using the microbial composition of the present invention, a field test is conducted, and the crop is tomato, and the plant disease is bacterial wilt, and the test field is treated with a complete random design (CRD). When plowing the field, farmers apply the following various fertilizers to the field soil. The field experiment was divided into three groups, namely the control group, the mixed organic fertilizer group and the miscellaneous organic fertilizer group. The control group is a base fertilizer that uses a mixture of organic fertilizers and chemical fertilizers at a rate of 200kg/slot; the mixed organic fertilizer group is a base fertilizer that uses organic fertilizers at a rate of 30-70kg/seat and a microbial composition of 130-170kg/seat; The group is a base fertilizer using 30-70 kg/min of chemical fertilizer and 130-170 kg/min of microbial composition. After planting for 2 months, the natural occurrence of tomato bacterial wilt was investigated. See figure 7. The results show that the groups containing the microbial composition, that is, the mixed organic fertilizer group and the miscellaneous organic fertilizer group have a lower incidence of bacterial wilt disease, which means that the microbial composition of the present invention can increase the incidence of bacterial wilt at approximately the same amount of fertilizer. Tomato's control of bacterial wilt.

In another embodiment of the method of using the microbial composition of the present invention, the embodiment is a field test, the crop is loofah, the plant disease is wilt, and the number of investigations is 5 times, and the interval is one week. Same as the aforementioned field experiment, divided into a control group, a mixed organic fertilizer group and a miscellaneous organic fertilizer group. The results are shown in Figure 8, which shows that the group containing the microbial composition has a lower morbidity rate of wilt, which indicates that the microbial composition of the present invention can enhance the control of loofah against wilt under the same amount of fertilizer. .

In summary, the present invention provides a strain (HL_B03) for the prevention and control of plant diseases, a microbial composition and a method of use thereof, so as to achieve the purpose of improving the nutrient status of the soil and preventing and controlling plant diseases.

Although the present invention has specifically described the strains, microbial compositions and methods of use for the prevention and control of plant diseases of the present invention with the above examples, those skilled in the art to which the present invention belongs should understand that they can be used without departing from the technical principles and methods of the present invention. In spirit, the embodiments are modified and changed. Therefore, the protection scope of the present invention should be as described in the scope of the patent application.

S1, S2, S3, S4, S5: steps

Figure 1 is a flow chart of the screening method of Bacillus strains of the present invention.

Figure 2 is a photograph of a strain that produces an antagonistic circle obtained by the screening method of the Bacillus strain of the present invention.

Figure 3 is the result of the experiment of inhibiting fungal spore germination of the strain of the present invention (Bacillus HL_B03) against Luffa wilt.

Figure 4 is a comparison diagram of the antagonistic effects of different sources of rapeseed meal in the microbial composition of the present invention.

Figure 5 is a comparison diagram of the antagonistic effects of the rapeseed meal water extracts of different dilution multiples of the present invention with the strain of the present invention against Ralstonia solanacearum.

Figure 6 is a diagram showing the results of an embodiment of the method of using the microbial composition of the present invention.

Figure 7 is a diagram showing the results of another embodiment of the method of using the microbial composition of the present invention.

Figure 8 is a diagram showing the results of another embodiment of the method of using the microbial composition of the present invention.

Domestic hosting information.

Food Industry Development Research Institute.

December 29, 2006.

Registration number: BCRC910810.

S1, S2, S3, S4, S5: steps

Claims (7)

A strain (HL_B03) of Bacillus velezensis , which is deposited at the Food Industry Development Institute of a consortium legal person, and the deposit label is BCRC910810, wherein the strain can antagonize bacterial wilt, and the strain adopts a screening method The screening method includes: providing soil containing the bacillus to a medium; culturing at 30°C for 24 to 48 hours, adding 108cfu/ml of bacterial wilt in the medium; selecting the antagonistic circle with a radius close to or greater than An initial strain of 1 cm; the initial strain is cultivated in a medium containing rapeseed meal (rapeseed meal) and cultured at 30° C. for 24 to 48 hours; and strains that can produce utilization circles are selected. A microbial composition for the prevention and control of plant diseases, comprising: a Bacillus strain (HL_B03); and a rapeseed meal; wherein the proportion of the strain in the microbial composition is 2 to 3%, and the rapeseed meal is The proportion in the microbial composition is 97-98%. The microbial composition according to claim 2, wherein the rapeseed meal includes at least one of mustard powder, rapeseed meal, and canola meal. The microbial composition according to claim 2, wherein the plant disease includes bacterial wilt or loofah wilt. The microbial composition according to claim 2, further comprising a base fertilizer, wherein the weight ratio of the microbial composition to the base fertilizer is 3:5-9:10. A method of using a microbial composition, comprising: applying the microbial composition as described in claim 2 to the soil where a crop is scheduled to be planted; or applying the microbial composition as described in claim 2 on the surface of the crop. The method of use according to claim 6, wherein the crops include Solanaceae or Cucurbitaceae crops.

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