CN110511893B - Bacillus biocontrol strain and application thereof - Google Patents

Bacillus biocontrol strain and application thereof Download PDF

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CN110511893B
CN110511893B CN201910809905.6A CN201910809905A CN110511893B CN 110511893 B CN110511893 B CN 110511893B CN 201910809905 A CN201910809905 A CN 201910809905A CN 110511893 B CN110511893 B CN 110511893B
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cyclocarya paliurus
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王海华
陶宗
彭喜旭
申权
高健
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Abstract

The invention discloses a bacillus biocontrol strain and application thereof. The strain is classified and named as Bacillus sp.QJK-3, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019377. Experiments show that: the Bacillus (Bacillus sp.QJK-3) has good biological control effect on cyclocarya paliurus leaf blight, and the control effect can reach 69.1%. The method lays a foundation for biological control of cyclocarya paliurus leaf blight, can reduce harm of chemical pesticides to environmental ecology and human health, and can improve and promote artificial cultivation, income increase and yield increase of cyclocarya paliurus.

Description

Bacillus biocontrol strain and application thereof
Technical Field
The invention relates to the field of biotechnology and microorganisms, and particularly relates to Bacillus (Bacillus sp.) QJK-3 for efficiently preventing and treating cyclocarya paliurus leaf blight and application thereof.
Background
Cyclocarya paliurus (Bata1.) Iljinskaja is a unique single-genus arbor of Cyclocarya paliurus (Bata1.) belonging to Juglauiaceae (Juglaceae) in China, is mainly distributed in mountainous areas with elevation of 420-2500 m in southern China, belongs to endangered rare plants, and has high medicinal, timber and ornamental values. The tea leaves have unique sweet taste, and local residents have custom for making tea for drinking, which is commonly called sweet tea trees. The cyclocarya paliurus contains various pharmacological components including polysaccharide, triterpene, saponin, flavonoid and the like, has various biological activities of reducing blood sugar, blood pressure and blood fat, resisting oxidation, diminishing inflammation and the like, and particularly has obvious auxiliary curative effect on diabetes.
The market has vigorous demands on raw materials and products of cyclocarya paliurus, is limited by the shortage of natural resources, and the artificial breeding of the cyclocarya paliurus is started recently. Leaf blight is an important disease of cyclocarya paliurus seedlings, is caused by Phomopsis eutropha (Phomopsis eutommiae), and mainly damages leaves and sometimes damages branches. The disease spots are initially dark brown or black small spots, then are enlarged to be nearly circular or irregular, gray black, the juncture of disease and health is not obvious, the disease spots are expanded and connected at the later stage, the whole plant is gray to black, and the plant can die when the disease spots are serious. The application of the chemical bactericide is still an effective means for preventing and treating cyclocarya paliurus leaf blight at present. However, the drawbacks of chemical fungicides are evident: high residue, easy generation of drug resistance, harm to non-target organisms, environmental pollution and ecological harm, and adverse effects on green environment-friendly pollution-free crop planting and sustainable development of agriculture, so that a safe and effective alternative measure for preventing and treating cyclocarya paliurus leaf blight is needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bacillus biocontrol strain and an application thereof, the bacillus has a strong antagonistic effect on cyclocarya paliurus leaf blight bacteria, the bacteriostasis rate can reach 72.2%, and the control effect can reach 69.1%.
The Bacillus biocontrol strain is classified and named as Bacillus sp.QJK-3, is preserved in China Center for Type Culture Collection (CCTCC) for short, is preserved in China, Wuhan university with the preservation number of CCTCC M2019377, and has the preservation date of 2019, 5 months and 20 days.
The Bacillus (Bacillus sp. QJK-3) is obtained by separating from cyclocarya paliurus rhizosphere soil, screening by adopting a plate confrontation method, and is identified as the Bacillus by colony morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequence determination and phylogenetic tree analysis.
The Bacillus (Bacillus sp. QJK-3) colony grows to the periphery in a random concave-convex manner, the edge is not smooth and is corrugated, and the surface has fold lines and is opaque and light yellow.
The physiological and biochemical characteristics of the Bacillus (Bacillus sp. QJK-3) are as follows: gram stain, oxidase, catalase, urease and nitrate reduction test positive; the esterase test, the methyl Red test, the V-P test and the indole production test were negative and did not produce H2S; d-glucose, D-raffinose and glycerol can be well utilized, D-xylose, inositol, L-rhamnose, L-arabinose, D-galactose and alpha-lactose cannot be utilized, and an inorganic nitrogen source, L-tyrosine and DL-a-alanine can be utilized; can liquefy gelatin, peptonize milk, hydrolyze casein, utilize citrate, and can not hydrolyze starch and cellulose.
The sequence similarity of the Bacillus (Bacillus sp. QJK-3) of the invention with a plurality of model strains such as B.velezensis CR-502T, Siamese Bacillus KCTC 13613T, Bacillus subtilis subsp.subtilis NCIB 3610T, Bacillus amyloliquefaciens DSM 7T and the like is more than or equal to 97 percent through 16S rDNA sequence comparison (EzBioCloud database).
The bacillus biocontrol strain can be used as a biological pesticide and applied to control cyclocarya paliurus leaf blight, and the control effect can reach 69.1%.
The invention has the beneficial effects that:
(1) the Bacillus (Bacillus sp.QJK-3) provided by the invention is a beneficial microorganism at the rhizosphere of cyclocarya paliurus, can be colonized on the body surface and in the body of the cyclocarya paliurus, can generate an active metabolite having an inhibiting effect on cyclocarya paliurus leaf blight bacteria by the strain and a fermentation liquid thereof, and has an obvious biological control effect on the cyclocarya paliurus leaf blight.
(2) The strain culture conditions are simple, the preservation is easy, the industrial production is easy, and the application prospect is good.
Drawings
FIG. 1 Single colony morphology of Bacillus sp.QJK-3 of the present invention on LB medium.
FIG. 2 phylogenetic tree (based on 16S rDNA sequence) of Bacillus sp.QJK-3 according to an embodiment of the present invention.
FIG. 3 shows the inhibition effect of Bacillus sp.QJK-3 on the growth of bacterial filaments of cyclocarya paliurus leaf blight.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.
Example 1 isolation and purification of Bacillus sp. QJK-3
Collecting a plurality of cyclocarya paliurus rhizosphere soil samples, weighing 1g of air-dried soil sample by adopting a continuous dilution method and a flat plate coating method, putting the air-dried soil sample into a conical flask filled with 100mL of sterile water, sealing a sterile sealing film, repeatedly oscillating to uniformly mix the soil sample, and standing for 30 min. Sequentially diluting the suspension into suspension by using a sterile pipette10-3、10-4、10-5、10-6、10-7And 10-8And (3) sucking 500 mu L of diluent respectively at six concentration gradients, uniformly coating the diluent on LB culture medium plates, placing the plates with three gradients in an incubator at 28 ℃ for 5 days, and selecting a single colony on the culture medium to perform streaking purification for more than three times. The single colony morphology of QJK-3 strain on LB medium is shown in FIG. 1.
The LB culture medium comprises the following components: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 1000mL of distilled water and pH 7.2-7.4.
Example 2 identification of Bacillus (Bacillus sp. QJK-3)
1. Morphological identification
The single colony morphology of the QJK-3 strain after incubation on LB medium at 37 ℃ for 1 day is shown in FIG. 1. FIG. 1 shows that its colony grows to the periphery irregularly and convexly, the edge is not smooth and is corrugated, and its surface has fold lines, and is opaque and light yellow.
2. Physiological and biochemical identification
QJK-3 Strain positive in gram stain, oxidase, catalase, urease, and nitrate reduction tests; esterase test, methyl Red test, V-P test and indole production test were negative and did not produce H2S; gelatin liquefaction, peptonization, casein hydrolysis, utilization of citrate, and inability to hydrolyze starch and cellulose (as shown in table 1).
TABLE 1 physiological and biochemical identification of Bacillus sp.QJK-3
Figure GDA0003583660790000031
QJK-3 strains unique carbon and nitrogen source utilization as shown in Table 2. QJK-3 strain can utilize D-glucose, D-raffinose and glycerol well, and can not utilize D-xylose, inositol, L-rhamnose, L-arabinose, D-galactose and alpha-lactose; inorganic nitrogen sources, L-tyrosine and DL-a-alanine can be utilized.
TABLE 2 utilization of carbon and nitrogen sources by Bacillus sp.QJK-3
Figure GDA0003583660790000032
Note: "+ +" indicates better growth; "+" indicates that there was growth but less bacteria; "-" indicates no growth.
3. Molecular identification
Shaking and culturing QJK-3 strain single colony in LB liquid culture medium for 24h (37 deg.C, 150rpm), centrifuging at 12000rpm for 1min, removing supernatant, and collecting thallus. The operation was carried out by using a bacterial genome DNA extraction kit (Biotechnology engineering (Shanghai) Co., Ltd.) according to the instructions. PCR was performed using genomic DNA of QJK-3 strain as a template, based on the prokaryotic microorganism 16SrDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). 100 μ l PCR reaction: 10 μ l of 10 XPCR buffer (containing MgCl)2) 2. mu.l of 10mmol/L dNTP, 5. mu.l of 10. mu. mol/L27F primer, 5. mu.l of 10. mu. mol/L1492R primer, 5. mu.l of template DNA (200ng), 2.5U Taq polymerase (Biotechnology engineering (Shanghai) Co., Ltd.), plus ddH2And (3) after the mixture is uniformly mixed to the final volume, carrying out amplification on a PCR instrument: after denaturation at 95 ℃ for 4min, PCR cycles are carried out, wherein each cycle comprises 30 cycles of 94 ℃ for 30s, 50 ℃ for 30s and 72 ℃ for 1 min; then extended for 7min at 72 ℃. The PCR amplification results were analyzed by detection on a 1% agarose gel electrophoresis (1% is the mass percentage of agarose). The PCR product is recovered by using a recovery kit and sent to biological engineering (Shanghai) company Limited for sequence determination. The sequencing result is spliced to obtain the 16SrRNA gene sequence of the QJK-3 strain, the length is 1419bp, and the NCBI (https:// www.ncbi.nlm.nih.gov /) accession number is MH 553055.
The homologous sequence search of the ezBioCloud database shows that the 16S rDNA sequence similarity of the QJK-3 strain and a plurality of model strains such as Bacillus belgii (B.velezensis CR-502T), Bacillus siamensis (B.siamensis KCTC 13613T), Bacillus subtilis subsp.subtilis NCIB 3610T and Bacillus amyloliquefaciens (B.amyloliquefaciens DSM 7T) is more than or equal to 97 percent. Coli 16S rRNA sequence as an ectogroup, and that the phylogenetic tree of the QJK-3 strain was established, QJK-3 strain and the strain of BacillusUnder the same large branch and with the model strain Bacillus amyloliquefaciens (B. amyloliquefaciens DSM 7)T) Clustering into the same branch (as shown in fig. 2). Combining the thallus morphology, the colony characteristics and the physiological and biochemical characteristics to identify the bacillus.
Example 3 antagonistic analysis of Bacillus (Bacillus sp. QJK-3)
The antagonistic effect of the QJK-3 strain on the growth of bacterial filaments of the cyclocarya paliurus leaf blight fungus is determined by adopting a plate opposing method. QJK-3 strain and cyclocarya paliurus (Phytopsis eutomiae) strain CB3 are reactivated, a sterile puncher (7mm) is used for punching, a pathogen agar block is clamped by sterile tweezers and placed in the center of a PDA (personal digital assistant) plate, a QJK-3 strain agar block is placed at a position 3cm away from the center of the plate, the inoculated sterile agar block is used as a control, the culture is carried out at 28 ℃ until the pathogen grows over the whole plate, the bacteriostasis effect is observed, and the bacteriostasis rate is calculated.
The bacteriostatic rate (%) is (Rp-Rt)/Rp multiplied by 100%
Wherein Rp represents the growth radius of the hypha of the control group, Rt represents the growth radius of the hypha of the treatment group (close to the antagonistic bacteria side), and the growth diameter of the hypha is calculated by subtracting the radius of the fungus cake (3.5mm) from the measured average value.
The cyclocarya paliurus leaf blight strain CB3 is Phomopsis eucommiae (Phomopsis eutomiae), is separated and identified from cyclocarya paliurus diseased leaves infected with leaf blight by an inventor, and is determined to be pathogenic bacteria.
The PDA culture medium comprises the following components: 200g potato, 20g glucose, 16g agar, 1000mL distilled water, natural pH.
The experimental result shows that the Bacillus (Bacillus sp.QJK-3) has a remarkable inhibition effect on the hypha growth of the cyclocarya paliurus leaf blight strain CB3 (as shown in figure 3), and the inhibition rate is 72.2%.
Example 4 greenhouse experiment of Bacillus sp.QJK-3 for controlling Black blight of cyclocarya paliurus
Selecting QJK-3 strain single colony in LB liquid culture medium, shake culturing for 24h (37 deg.C, 150rpm), preparing with sterile water to 1 × 107cfu/mL of bacterial suspension for use.
Inoculating cyclocarya paliurus leaf blight bacterium (Phomopsis eutomiae) CB3 strainCulturing on PDA culture medium at 25 deg.C for 22 days, collecting conidia, and preparing with sterile water to obtain 1 × 10 conidia6cfu/mL spore suspension. Taking soil in field, sun drying for 1-2 days, grinding, sieving, spraying spore suspension with common plastic sprinkling can, stirring thoroughly, making into soil with bacteria, and placing into ceramic basin with upper caliber of 60cm and bottom caliber of 45cm, wherein the soil loading amount is 2/3 of the volume of the ceramic basin. Planting healthy cyclocarya paliurus seedlings of 1 age in each pot, and culturing in an outdoor greenhouse. The culture conditions are as follows: temperature 25-28 deg.c (day)/20-23 deg.c (night), relative humidity 85-90% and natural light period. The following 3 treatments were used: (1) clear water control; (2) bacillus QJK-3 strain suspension (1X 10)8cfu/mL); (3) thiophanate-methyl (70% by weight of wettable powder, one of chemical bactericides). Dipping roots before planting cyclocarya paliurus seedlings, and fully soaking the roots in the 3 treatment liquids for 20min respectively. Spraying the plants with the 3 treatment solutions for 30d to moisten the plants, and examining the disease condition 60d after spraying.
The disease grade standard is as follows: grade 0, no leaf blight symptom; grade 1, with 1-25% of the leaves showing symptoms of leaf blight; grade 2, with black leaf withered symptoms appearing in 26-50% of leaves; grade 3, with black leaf withered symptoms appearing in 51-75% of leaves; over 76% of the leaves exhibited a black leaf-withered symptom. Disease index ∑ [ number of diseased leaves at each level × relative disease value at each level ]/[ number of investigated total leaves ] × highest disease value. The control effect (%) is (control disease index-treatment disease index)/control disease index x 100.
As shown in Table 3, the control effect of the bacillus QJK-3 strain on the cyclocarya paliurus leaf blight reaches 69.1%, and the control effect is not obviously different from that of thiophanate methyl (P > 0.05).
TABLE 3 greenhouse control of cyclocarya paliurus leaf blight by Bacillus QJK-3
Figure GDA0003583660790000051

Claims (2)

1. A Bacillus biocontrol strain is characterized in that the Bacillus biocontrol strain is classified and named as Bacillus sp.QJK-3, is preserved in China Center for Type Culture Collection (CCTCC) for short, is preserved in China, Wuhan university and has the preservation number of CCTCC NO: m2019377, with a preservation date of 2019, 5 months and 20 days.
2. The application of the bacillus biocontrol strain in preventing and treating cyclocarya paliurus leaf blight disease as claimed in claim 1, wherein the cyclocarya paliurus leaf blight disease is caused by phomopsis cortex.
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