CN115948247B - Chinese secretoglycete and application thereof - Google Patents
Chinese secretoglycete and application thereof Download PDFInfo
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Abstract
The application discloses a Chinese secretoglycete and application thereof, and relates to the technical field of plant pathogenic bacteria control. The said Chinese darkball is named Chinese darkball (Phaeosphaeria sinensis), strain ZJG2022PS, preservation number is CCTCC NO: m2022716. The Chinese dark-ball chamber bacterium ZJG2022PS provided by the application has the advantages that the growth inhibition rate of hypha growth of the citrus black-spot bacterium can reach 36% in a plate counter test, the inhibition effect of the hypha growth inhibition rate of the citrus black-spot bacterium can reach 27.78% in a fermentation liquor test, and the strain ZJG2022PS can effectively inhibit the growth of the citrus black-spot bacterium, can be used for preparing a novel, efficient and broad-spectrum biocontrol microbial agent, and provides a new way for the application of microorganisms in the development of biological control.
Description
Technical Field
The application relates to the technical field of plant pathogenic bacteria control, in particular to a Chinese trichosphaerella and application thereof.
Background
Citrus is an important agricultural crop, and its safe production is of great importance. Pathogenic bacteria which harm oranges are various, and the orange black spot bacteria are one of the pathogenic bacteria. Citrus black spot seriously affects the quality of citrus and causes great economic loss to fruit farmers. Citrus black spot disease generally occurs in young fruits, young shoots and young leaves of citrus, innumerable brown, blackish brown scattered or densely-flaked hard gum small particles are generated on the surface of the diseased part, the appearance quality of citrus is seriously affected, the quality of fruits is seriously reduced, and huge economic loss is caused. In addition, the citrus black spot germs can also carry out saprophytic growth on plant tissue residues and form a large number of fruit bodies, wherein dead branches with germs in an orchard are main infection sources of the citrus black spot germs.
Biological control refers to a method for controlling plant diseases and insect pests by using organisms and metabolites thereof. The essence of the method is to utilize the relationship between organism species and the relationship in the species to regulate the population density of harmful organisms, namely the biological group prevention and control biological group. Compared with the traditional control technology, the biological control technology has the advantages of no environmental pollution, safety to people and other organisms, no residue of products, easy coordination with other plant protection measures, energy conservation and the like, and plays an increasingly important role in the comprehensive control of harmful organisms. Fungi, bacteria, actinomycetes and yeasts have been used for biological control to date. The use of bacteria to control harmful microorganisms is one of important research fields for developing and utilizing microbial resources, and is also one of the most effective ways with the highest potential and the widest application prospect. The bacterial control advantages mainly include the following points: firstly, the breeding speed is high, and the culture cost is low; secondly, the application modes of the microbial inoculum on pathogenic bacteria are wider, such as bacteriocins, antibiotics, extracellular lyase, lipopeptides and other various forms; thirdly, some bacteria have the dual functions of preventing diseases and promoting growth.
The patent application with publication number of CN109221152A discloses a bactericidal pesticide composition containing active ingredients of fluxapyroxad and thiabendazole, wherein the weight ratio of the fluxapyroxad to the thiabendazole is 1:49 to 49:1. the composition can be prepared into various pesticide preparations, and can be used for preventing and treating various bacterial and fungal diseases of agricultural and forestry crops such as fruit trees, forests, vegetables, grain crops and the like.
The literature Chen Guoqing and the like discloses the inhibition effect of 3 ergosterol synthesis inhibitor bactericides (tebuconazole, difenoconazole, prochloraz manganese chloride) and mancozeb on the hypha growth and conidium germination of the black spot pathogen under laboratory conditions in the in vitro and field screening of medicines for preventing and treating the black spot disease of citrus, zhejiang university journal (agricultural and life science edition) and 2010 period 04. The results show that: the effective medium concentration (EC 50) of the 4 medicaments for inhibiting the growth of the hyphae of the black spot bacteria is 0.250, 0.497, 0.113 and 1.800 mug/mL in sequence; the EC50 for inhibiting spore germination was 72.893, 42.746, 20.701 and 0.970 μg/mL, in that order. The field prevention test of the medicament shows that: the 600-time diluent of the mancozeb wettable powder has the prevention and treatment effects on the black spot disease of 79.1 percent and 63.6 percent respectively after being used for 5 times and 3 times, and is obviously superior to the bactericides of tebuconazole, difenoconazole and prochloraz manganese of the same times and recommended dosage. Therefore, mancozeb is considered to be the best agent for controlling citrus black spot among these 4 agents.
In the actual production process, traditional pesticides are mostly used for prevention and control. It is reported in literature that the pesticide utilization is generally 10%, and about 90% of the pesticide remains in the environment, causing environmental pollution. In addition, pesticide residues can also cause harm to people and livestock. Therefore, the screening of beneficial fungi for biological control is an important method for improving the safety production of citrus.
Disclosure of Invention
The application provides a kind of Chinese darkling globus and its application, the strain ZJG2022PS of the application is separated from the soil of Xuan Wu Ou purple gold mountain Bo garden (North latitude 32.048687 degree, east longitude 118.797861 degree) in Nanjing, jiangsu province. Through sequence comparison, the ZJG2022PS strain is closest to the sequence of the Proteus scoliosis (Phaeosphaeria sinensis), and the result is the same as the morphological identification result, which shows that the ZJG2022PS strain isolated by us is the Proteus scoliosis (Phaeosphaeria sinensis).
The application provides a kind of Chinese darkball chamber fungus, named Chinese darkball chamber fungus (Phaeosphaeria sinensis), strain ZJG2022PS, preservation number CCTCC NO: m2022716.
The application also provides application of the Chinese dark-bulb cavity bacteria in inhibiting growth of citrus black spot bacteria (Phoma citricarpa McAlp.).
The application also provides application of the Chinese dark-bulb fungus in preventing and treating infection of citrus black spot fungus (Phoma citricarpa McAlp.) of Rutaceae plants.
All plants which can infect the citrus black spot bacteria can be used. In addition to the citrus, other plants of the family Rutaceae such as orange, grapefruit, etc. may be used. Preferably, the Rutaceae plant is a citrus, orange, pomelo, lemon or orange.
The application also provides a citrus black spot germ inhibitor, and the active ingredient comprises the Chinese trichosphaerella.
The application also provides a preparation method of the citrus black spot bacteria inhibitor, and after the Chinese trichomonas is inoculated into a fermentation medium for culture, the fermentation liquid from which the thalli are removed is the citrus black spot bacteria inhibitor.
Preferably, the incubation temperature is 25℃and the incubation time is 3 days.
The application also provides a control method of the citrus black spot bacteria, and the citrus black spot bacteria inhibitor or the Chinese trichosphaerella is used for being sprayed on plants. Can be used for preventing and treating plants infected with the black spot disease of citrus.
The application has the beneficial effects that:
the Chinese dark-ball chamber bacterium ZJG2022PS provided by the application has the advantages that the growth inhibition rate of hypha growth of the citrus black-spot bacterium can reach 36% in a plate counter test, the inhibition effect of the hypha growth inhibition rate of the citrus black-spot bacterium can reach 27.78% in a fermentation liquor test, and the strain ZJG2022PS can effectively inhibit the growth of the citrus black-spot bacterium, can be used for preparing a novel, efficient and broad-spectrum biocontrol microbial agent, and provides a new way for the application of microorganisms in the development of biological control.
Drawings
FIG. 1 is a colony morphology of the Chinese Chaetoceros ZJG2022PS grown on CM medium for 6 days; wherein A is a front view of the culture dish, and B is a back view of the culture dish.
FIG. 2 is a diagram showing the detection result of the inhibition of the Chinese Cavity surface bacteria ZJG2022PS to the citrus black spot bacteria; wherein A is a graph of inhibition of the Chinese trichomonas ZJG2022PS on the citrus black spot bacteria, and B is the growth condition of the control Check (CK) citrus black spot bacteria.
FIG. 3 is a graph and a line graph showing the inhibition effect of the fermentation broth of the Caesalpinia chinensis ZJG2022PS on the black spot bacteria; wherein A is a graph for detecting the inhibiting effect of the Chinese trichomonas ZJG2022PS fermentation liquor on the black spot bacteria, and B is a graph for detecting the inhibiting effect of the Chinese trichomonas ZJG2022PS fermentation liquor on the black spot bacteria.
Detailed Description
Example 1: bacterial separation
(1) Soil samples were collected from the Xuan Wu Ou African mountain Bo garden in Nanjing, jiangsu province and separated by a selective medium dilution plate method, the process was as follows: taking 5 centrifuge tubes of 1.5mL, marking with a marker pen with 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Pipette 0.9mL of sterile water into each centrifuge tube;
(2) 1g of the sample is weighed and put into a test tube with a plug filled with 9mL of sterile water, and the sample is oscillated for 10min to obtain 10 -1 Is a soil dilution of (2);
(3)10 -1 standing for 2min after shaking the soil diluent, sucking 0.1mL of suspension by a pipette, adding into a centrifuge tube filled with 0.9mL of sterile water, and sequentially diluting to obtain 10 -2 ，10 -3 、10 -4 、10 -5 、10 -6 A dilution liquid;
(4) CM medium (with streptomycin and ampicillin added to the medium to make it reach 30 μg/L to inhibit bacterial growth) cooled to 50-60deg.C after sterilization is poured into culture dishes with 20mL each; after the culture medium is cooled to be flat, respectively sucking 10 by using a pipettor -6 、10 -5 、10 -4 、10 -3 Adding 0.1mL of the suspension of the diluent into a flat plate, and uniformly coating each diluent by using a sterile coating rod, wherein each concentration is repeated by 3 dishes;
(5) The culture dish is inversely cultured in a constant temperature incubator for 3-5 days at 25 ℃ and then mycelium of single colony is picked on a new flat plate.
Table 1 CM reagents and amounts (1L) used in the Medium
| Reagents used | Dosage of |
| D-glucose (D-glucose) | 10g |
| Peptone (Peptone 140) | 2g |
| Casein extract (Casamino acid) | 1g |
| Yeast extract (Yeast extract) | 1g |
| Sodium nitrate (NaNO) 3 ) | 6g |
| Monopotassium phosphate (KH) 2 PO 4 ) | 1.52g |
| Potassium chloride (KCl) | 0.52g |
| Magnesium sulfate heptahydrate (MgSO) 4 ·7H 2 O) | 0.52g |
| Vitamin solution (1000 XVitamin solution) | 1mL |
| Microelements (1000X trace elements) | 1mL |
The above reagents are weighed according to the corresponding amounts, and then the volume is fixed to the corresponding volume, wherein the formula of the vitamin solution is shown in table 2, and the formula of the trace elements is shown in table 3. The pH was adjusted to 6.5 with NaOH, 15g of agar powder per liter of the solid medium was added, and the medium was autoclaved at 121℃for 15min.
TABLE 2 1000 XVitamin solution (100 mL)
| Reagents used | Weight/gram |
| Biotin (Biotin) | 0.01g |
| Vitamin B (Pyridoxin) | 0.01g |
| Thiamine (Riboflavin) | 0.01g |
| Riboflavin (Thiamine) | 0.01g |
| Nicotinic acid (Nicotinic acid) | 0.01g |
| Para aminobenzoic acid (p-aminobenzonic acid) | 0.01g |
The reagent is dissolved in distilled water and then stored in a refrigerator at 4 ℃ until the volume reaches 100 mL.
TABLE 3 1000 XTrace Elements (100 mL)
| Reagents used | Weight/gram |
| Sodium molybdate pentahydrate (Na) 2 MoO 4 ·5H 2 O) | 0.15g |
| Pentahydrate copper sulfate (CuSO) 4 ·5H 2 O) | 0.16g |
| Cobalt chloride hexahydrate (CoCl) 2 ·6H 2 O) | 0.17g |
| Manganese chloride tetrahydrate (MnCl) 2 ·4H 2 O) | 0.5g |
| Ferrous sulfate heptahydrate (FeSO) 4 ·7H 2 O) | 0.5g |
| Boric acid (H) 3 BO 3 ) | 1.1g |
| Zinc sulfate heptahydrate (ZnSO) 4 ·7H 2 O) | 2.2g |
| Ethylene diamine tetraacetic acid tetrasodium salt (Na 4 EDTA) | 5g |
Dissolving the above reagents in distilled water, and keeping volume to 100mL, and storing in a refrigerator at 4deg.C in dark place.
YG medium: 10g glucose, 5g yeast extract, 15g agar, 1000mL distilled water.
A strain was isolated from soil of the Xuan Wu Ou African mountain Bo garden (32.048687 degrees North latitude, 118.797861 degrees east longitude) in Nanjing, jiangsu province and named ZJG2022PS.
Example 2: identification of strain morphology
Colony morphology characteristics of CM medium were categorized.
Bacterial strain ZJG2022PS has dense aerial hyphae and slightly diverges on CM culture medium; culturing at 25deg.C under 16 hr light for 8 hr dark condition alternately, wherein the average growth rate of mycelium is 4.53-4.62mm per day, and the mycelium can grow into plate with diameter of 90mm after 19.48-19.88 d. The hyphae spread circularly, with the front side being substantially greenish black (fig. 1A) and the back side being earthy yellow (fig. 1B). And combining with the molecular biology identification result, identifying the strain ZJG2022PS as the Chaetomium globosum.
Example 3: molecular characterization
(1) DNA extraction
1) After culturing ZJG2022PS strain on CM plate at 25deg.C for 7 days, scraping hypha on the plate with toothpick, placing into sterilized 1.5mL centrifuge tube containing 500 μl extraction buffer and appropriate amount of quartz sand;
the formula of the extraction buffer is as follows: 10mM ethylenediamine tetraacetic acid, 100mM Tris-hydrochloric acid, 1M potassium chloride;
2) Place centrifuge tube in MP-24 homogenizing and crushing the mycelium tissue by shaking for 2min at 65 Hz;
3) Centrifuge 12000rpm,10min, pipette 400 μl supernatant and pour into a fresh centrifuge tube;
4) Adding isopropanol with the same volume as the supernatant, mixing the mixture upside down, placing the centrifuge tube in a refrigerator at 4 ℃ for precipitating DNA better, and precipitating at low temperature for 10min;
5) Centrifuging at 12000rpm for 10min, pouring out supernatant, and retaining precipitate;
6) Adding 70% ethanol (v/v) 800 μl to dissolve impurities, gently mixing, and centrifuging at 12000rpm for 2min;
7) Discarding the supernatant, placing the centrifuge tube on absorbent paper in a reverse way to volatilize redundant ethanol, adding 50 mu L of sterile water into the white precipitate to obtain the genome DNA to be extracted, dissolving the genome DNA for 15min at room temperature, and placing the genome in a refrigerator at-20 ℃ for preservation after the dissolution is finished.
(2) PCR amplification of fungal ribosomal ITS rDNA Gene and ribosomal LSU rDNA Gene
ITS primer: the sequence of the upstream primer ITS1 is as follows: 5'-TCCGTAGGTGAACCTGCGG-3', the downstream primer ITS4 sequence is: 5'-TCCTCCGCTTATTGATATGC-3';
LSU primer: upstream primer LR5: ATCCTGAGGGAAACTTC, downstream primer LROR: ACCCGCTGAACTTAAGC;
the PCR amplification was performed in a 25. Mu.L reaction system comprising: 1 mu L of each of the upstream and downstream primers, 12.5 mu L of Green Taq Mix, 1 mu L of template DNA and ddH 2 O 9.5μL。
The PCR amplification reaction was performed on a Langmuir MG96G type PCR apparatus. Reaction conditions: pre-denaturation at 94 ℃ for 4min, then 35 cycles included: denaturation at 94℃for 40s, annealing at 55℃for 50s, extension at 72℃for 1min; finally, the mixture is extended for 10min at 72 ℃.
(3) Sequencing and sequence analysis of genes
The DNA fragment of interest was sent to Nanjing general Biotechnology (Anhui) Inc. for sequencing. The sequencing result is strictly checked to obtain the DNA fragment sequences shown as SEQ ID No. 1-2, namely the PCR amplified products of the fungal ribosomal ITS rDNA gene (shown as SEQ ID No. 1) and the PCR amplified products of the ribosomal LSU rDNA gene (shown as SEQ ID No. 2).
The ITS nucleotide sequence and LSU gene sequence of the ZJG2022PS strain were determined on NCBI website and the homologous or similar nucleotide sequences were searched and aligned in the GenBank database using BLAST. The comparison shows that the sequence has 100% similarity with Phaeosphaeria sinensis of accession number MN 173213. Through sequence comparison, the ZJG2022PS strain is closest to the sequence of the Proteus scoliosis (Phaeosphaeria sinensis), and the result is the same as the morphological identification result, which shows that the ZJG2022PS strain isolated by us is the Proteus scoliosis (Phaeosphaeria sinensis). The newly selected strain is named as the Chinese darkling globus (Phaeosphaeria sinensis), the strain number is ZJG2022PS, and the strain is preserved in China center for type culture collection (CCTCC NO) at university of Wuhan in 2022, 5 months and 25 days: m2022716.
Example 4: inhibition of ZJG2022PS strain on citrus black spot bacteria
The ZJG2022PS strain was cultivated against the black spot pathogen (the black spot pathogen strain is given away by the university of Zhejiang Wang Hongkai teacher): on a CM plate with a diameter of 90mm, black spot orange bacteria and ZJG2022PS strain were inoculated on both sides of the plate, respectively, with a distance of 5CM between the two strains. After the ZJG2022PS strain and the citrus black spot bacteria are cultivated on the CM flat plate in a counter manner for 6d, the growth of the front edge of the colony of the citrus black spot bacteria is inhibited in the direction close to ZJG2022PS strain, and hypha is in a significantly inhibited state (figure 2), and the hypha growth inhibition rate can reach 36% (growth inhibition rate= (CK citrus black spot bacteria radius-counter-cultivated citrus black spot bacteria radius)/CK citrus black spot bacteria radius) through measurement and calculation.
Example 5: inhibition detection of ZJG2022PS strain on citrus black spot bacteria
ZJG2022PS strain is inoculated in 100mL CM culture medium/250 mL conical flask, cultured for 3d at 25 ℃ at 150rpm, the supernatant is obtained by centrifugation to obtain fermentation broth, the fermentation broth is filtered and sterilized, and then the fermentation broth is added into YG culture medium according to a proportion to prepare a fermentation broth-YG flat plate, and then citrus black spot bacteria are inoculated, and the antibacterial effect is observed for 4 days. The citrus black spot germs were inoculated on YG medium containing 5%, 10%, 20% and 30% of fermentation broth (v/m) and cultured for 4 days, respectively, and three replicates were set at different concentrations in each group using YG medium containing 5%, 10%, 20% and 30% of sterile water (v/m) as a control, respectively. As shown in FIG. 3A, the higher the concentration of the fermentation liquid, the more remarkable the inhibition effect on the citrus black spot bacteria, while the control group had no inhibition effect on the citrus black spot bacteria. The inhibition effect is 3.91% when 5% fermentation broth is added, as shown in the line diagram of FIG. 3B. When 30% of fermentation liquor is added, the inhibition effect can basically reach 27.78%.
Claims (5)
1. The Chinese trichothecene is characterized by being named as Chinese trichothecene (Phaeosphaeria sinensis), and has a strain number of ZJG2022PS and a preservation number of CCTCC NO: m2022716.
2. Use of the species darkling globus of claim 1 for inhibiting the growth of citrus black spot bacteria (Phoma citricarpa mcalp.).
3. The use of the Chinese dark-bulb fungus according to claim 1 in preparing a black spot pathogen inhibitor, wherein the active ingredient of the black spot pathogen inhibitor comprises the fermentation broth after fermentation culture of the Chinese dark-bulb fungus according to claim 1.
4. The use according to claim 3, wherein the inhibitor of black spot bacteria of citrus is prepared by:
inoculating the Chinese dark bulb fungus of claim 1 into a fermentation medium for culturing, and removing the fermentation liquor of the fungus body to obtain the citrus black spot fungus inhibitor.
5. The method according to claim 4, wherein the cultivation is carried out at a temperature of 25℃for a period of 6 days.
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Citations (2)
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| JP2005073583A (en) * | 2003-09-01 | 2005-03-24 | Shizuoka Prefecture | New simplicillium microorganism having ability to control citrus disease injury and microbe-controlling method using the same |
| CN110982709A (en) * | 2019-12-25 | 2020-04-10 | 浙江大学 | Trichoderma asperellum capable of inhibiting growth of citrus alternaria alternata and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005073583A (en) * | 2003-09-01 | 2005-03-24 | Shizuoka Prefecture | New simplicillium microorganism having ability to control citrus disease injury and microbe-controlling method using the same |
| CN110982709A (en) * | 2019-12-25 | 2020-04-10 | 浙江大学 | Trichoderma asperellum capable of inhibiting growth of citrus alternaria alternata and application thereof |
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