CN117106639A - Streptomyces nojirimensis strain and application of fermentation liquor thereof in preventing and controlling pepper anthracnose - Google Patents
Streptomyces nojirimensis strain and application of fermentation liquor thereof in preventing and controlling pepper anthracnose Download PDFInfo
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- CN117106639A CN117106639A CN202310952404.XA CN202310952404A CN117106639A CN 117106639 A CN117106639 A CN 117106639A CN 202310952404 A CN202310952404 A CN 202310952404A CN 117106639 A CN117106639 A CN 117106639A
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- Prior art keywords
- streptomyces
- strain
- nojirimensis
- anthracnose
- nojiriensis
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention relates to a Streptomyces nojirimensis strain and application of fermentation liquor thereof in preventing and controlling pepper anthracnose, belonging to the technical field of crop disease control. Biocontrol strain 9-13, named Streptomyces nojirimensis, is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 22798, and is stored in 2021, 6 and 28 days. The invention has the advantages that: the Streptomyces nojirimensis strain 9-13 has antagonism on Sgavill anthracnose, colletotrichum glomeratum, boning anthracnose, karst anthracnose and C.aenigma which cause the anthracnose of the capsicum, the fermentation liquor can obviously inhibit the hypha growth and spore germination of the 6 capsicum anthracnose, and the fermentation liquor has excellent control effect on the capsicum anthracnose.
Description
Technical Field
The invention relates to the technical field of crop disease control, in particular to application of a Streptomyces nojirimensis strain and fermentation liquor thereof in controlling pepper anthracnose.
Background
The Capsicum (Capsicum annuum l.) is a annual or effective perennial herb of the genus Capsicum of the family solanaceae, and is one of the most important cash crops in our country.
However, the production of the capsicum is limited by a plurality of diseases, so that the yield is seriously reduced, and the economic benefit is reduced. Currently epidemic disease, viral disease and anthracnose become three major diseases that jeopardize the safe production of capsicum. Wherein, the pepper anthracnose is commonly generated worldwide, and can be harmful from the seedling stage to the adult plant stage, mainly harmful fruits, stems and leaves, fruit stalks and seedlings, leading to death of the seedlings, fallen leaves and rotten fruits.
Pepper anthracnose is a disease caused by anthrax (Colletotrichum Cord) fungi. The variety of the pepper anthracnose germs is multiple, the population genetics are complex, and the field compound infection is frequent. The control measures of the pepper anthracnose mainly comprise agricultural control and chemical control. Agricultural control mainly uses planting disease-resistant varieties. At present, the variety with high anthracnose resistance is identified to be Chinese capsicum and sagged capsicum, but is difficult to be directly applied to breeding due to interspecific hybridization obstacle.
Biological control has become a research hot spot gradually due to the advantages of environmental protection, long lasting period, environmental, ecological and human health safety and the like. Wherein actinomycetes can produce a plurality of antibacterial active substances such as antibiotics, plant hormones, hydrolytic enzymes and the like, and have the potential of developing biological source pesticides. The biocontrol actinomycetes are prepared into the viable cell preparation, so that the soil structure can be improved, the pesticide residue is reduced, the environment compatibility is good, and the lasting period is long. The streptomyces garcinia gray Streptomyces luteogriseus antibacterial active products are rich in variety, and people sequentially separate the antibacterial active products such as oligomycin A, C amino acid antibiotics KT-151 and the like from fermentation liquor, so that the streptomyces garcinia gray Streptomyces luteogriseus antibacterial active products have obvious inhibition effect on various diseases. Prisana et al in 2019 reported that Streptomyces angustmyceticus has excellent control effect on leaf spot of cabbage caused by anthrax and Curvularia lunata, can release volatile antifungal compounds including alcohols, aldehydes, carboxylic acids and fatty acids, and can also produce various cell wall degrading enzymes.
Biocontrol bacteria protect plants from various pathogens by producing antibiotics, volatile compounds, synthesizing extracellular enzymes that degrade fungal cell walls, inducing systemic resistance, competing for nutrients and niches, and the like. The biocontrol actinomycetes are prepared into the viable cell preparation, so that the soil structure can be improved, the pesticide residue is reduced, the environment compatibility is good, the duration is long, and the method has important significance for sustainable development of agriculture.
Disclosure of Invention
The invention aims at seriously impeding the healthy development of the capsicum industry in the world aiming at the occurrence of anthracnose and providing the application of the streptomyces nojirimis (Streptomyces nojiriensis) strain 9-13 in the control of capsicum anthracnose.
The invention further aims to provide application of the Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth in preventing and controlling pepper anthracnose.
The aim of the invention is realized by the following technical scheme:
application of Streptomyces nojirimis strain in preventing and treating pepper anthracnose, wherein the strain is Streptomyces nojirimis (Streptomyces nojiriensis) strain 9-13, and is preserved in China general microbiological culture Collection center (CGMCC) No.22798, and the preservation date is: 2021, 6, 28. Deposit unit address Beijing city, chaoyang area, north Chenxi Lu 1, 3
The strain 9-13 of the streptomyces nojirimi (Streptomyces nojiriensis) has strong antagonism on the Sgavill anthrax (Colletotrichum scovillei), the sclerotinia serrulata (C.fructicola), the colletotrichum truncatum (C.truncatum), the boninga anthracis (C.boninense), the karst anthrax (C.karst ti) and the C.aenigma which cause the pepper anthracis.
The application of the fermentation liquor of the strain of the streptomyces nojirimis in preventing and controlling pepper anthracnose is provided, wherein the fermentation liquor is prepared by inoculating 9-13 of the strain of the streptomyces nojirimis (Streptomyces nojiriensis) in an ISP2 liquid medium, fermenting at 26-28 ℃ for 7-8d, and filtering.
The strain 9-13 fermentation liquor of the streptomyces nojirimi (Streptomyces nojiriensis) has an inhibiting effect on hypha growth of the colletotrichum glomeratum (Colletotrichum scovillei), the colletotrichum glomeratum (C.fructicola), the colletotrichum glomeratum (C.truncatum), the colletotrichum boinicum (C.boninese), the karst colletotrichum (C.karst) and the C.aenigma.
The strain 9-13 fermentation liquor of the streptomyces nojirimi (Streptomyces nojiriensis) has an inhibiting effect on spore germination of colletotrichum glomeratum (Colletotrichum scovillei), colletotrichum glomeratum (C.fructicola), colletotrichum glomeratum (C.truncatum), colletotrichum boinium (C.boninense), karst colletotrichum (C.karst) and C.aenigma.
Compared with the prior art, the invention has the advantages that: the strain 9-13 of the nojirimycin (Streptomyces nojiriensis) has antagonism on 6 anthracnose bacteria causing the pepper anthracnose, the fermentation liquor can obviously inhibit the hypha growth and spore germination of the 6 pepper anthracnose bacteria, and the prepared fermentation liquor has excellent control effect on the pepper anthracnose.
In addition, the strain 9-13 of the Streptomyces nojirimensis (Streptomyces nojiriensis) is obtained from soil, is compatible with the soil environment in a harmonious manner, and has good application prospect.
Drawings
FIG. 1 shows the culture characteristics of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 on medium of Gao's first.
FIG. 2 shows the inhibitory effects of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 on Gaoker anthrax (Colletotrichum scovillei), cephalosporium kulardii (C.fructicola), cephalosporium crassifolium (C.truncatum), boning anthrax (C.boninense), C.aenigma and karst anthrax (C.karstii) under counter culture conditions. And (3) injection: a is GaoVil anthrax, B is Cephalosporium fulgicum, C is Cephalosporium crassifolium, D is Boning anthrax, E is C.aenigma, F is karst anthrax, and CK is control group.
FIG. 3 shows the inhibitory effect of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth on mycelial growth of Bacillus stearothermophilus (Colletotrichum scovillei), cellospora fruit (C.fructicola), cellospora crassa (C.truncatum), botrytis cinerea (C.boninense), C.aenigma and Zostertagia karst (C.karstini). And (3) injection: a is GaoVil anthrax, B is Cephalosporium fulgicum, C is Cephalosporium crassifolium, D is Boning anthrax, E is C.aenigma, F is karst anthrax, and CK is control group.
FIG. 4 shows the inhibitory effect of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth on spore germination of Leptospira stevensis (Colletotrichum scovillei), cephalosporium carpum (C.fructicola), cephalosporium acremonium (C.truncatum), boning anthrax (C.boninense), C.aenigma and Zostertagia karst (C.karst). And (3) injection: a is spore germination of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of Leptospira nojirimensis, B is spore germination of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of Leptospira rupestis, C is spore germination of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of Leptospira rupestis, D is spore germination of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of Boning anthrax, E is spore germination of Streptomyces nojirimis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of C.aenigma, F is spore germination of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth treated spore of Leptospira ruri, and CK is control group.
Detailed Description
The present invention is described in detail below with reference to the drawings and examples of the specification:
embodiment one: isolation and identification of Streptomyces nojirimoniae (Streptomyces nojiriensis) strain 9-13.
1. Soil sample collection
3 parts of soil samples are collected from Jiangxi Jinggang mountains, soil on the surface is removed, soil samples in the depth of 5-20cm are collected, and the soil samples are marked and then brought back to a laboratory for natural air drying.
2. Isolation of actinomycetes
The separation was performed by plate dilution. Grinding air-dried soil sample with mortar, suspending 1g of sample in 9mL of sterile water, shaking at 40deg.C and 180rpm for 30min, standing for 5min, sequentially diluting 10 times, and respectively preparing into 10 -2 、10 -3 、10 -4 Respectively sucking 0.1mL of each suspension with different concentrations, adding the suspension into a modified HVA culture medium (adding potassium dichromate with the final concentration of 100-200 ppm) plate, uniformly coating, then inverting the solution, culturing at 28 ℃, picking different single bacterial colonies after 5-7 days, streaking and purifying, and preserving the purified bacterial strains in a refrigerator at-80 ℃ by adopting a glycerol method.
3. Identification of Streptomyces nojirimensis (Streptomyces nojiriensis) Strain 9-13
(1) Morphological feature observations
Streptomyces nojirimoniae (Streptomyces nojiriensis) strain 9-13 grew well on most media and did not produce soluble pigments (Table 1). Under an optical microscope, the strain 9-13 has straight spore filaments, flexible filaments, hooks, loose filaments, tight spirals, oval spores and columns.
Table 1 culture characteristics of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13
(2) Physiological and biochemical characteristics
The characteristics of Streptomyces nojirimonia (Streptomyces nojiriensis) such as 9-13 nitrate reduction and carbon and nitrogen source utilization were measured by the method described in Streptomyces appraisal manual, and the results are shown in Table 2.
TABLE 2 physiological and biochemical characterization of strains 9-13
(3) Sequence analysis
After bacterial genome extraction kit extracts bacterial 9-13 genome DNA, respectively amplifying 16S rRNA and rpoB genes, obtaining sequences with the total length of 1409bp and 859bp, submitting the obtained sequences to GenBank database for BLAST comparison analysis, and obtaining bacterial strain 9-13 which is Streptomyces nojirimensis.
Embodiment two: antagonism of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 against pepper anthracnose pathogens was determined.
Antagonism of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 against Leptosphaeria spinosa, leptosphaeria crassifolia, leptosphaeria boinica, leptosphaeria clathrata and Leptosphaeria karst was determined by plate-counter culture. Firstly, inoculating Streptomyces nojirimensis (Streptomyces nojiriensis) strains 9-13 on two sides of the edge of a PDA culture medium by streaking, inoculating a tested pathogenic bacteria cake with the diameter of 5mm in the center of a flat plate after 3d, culturing for 5-7d at 28 ℃, and measuring the antibacterial zone width and the pathogenic bacteria diameter (Table 3) of the Streptomyces nojirimensis (Streptomyces nojiriensis) strains 9-13 on the tested pathogenic bacteria, wherein the tested pathogenic bacteria which are not connected with antagonistic bacteria are used as a control. The results showed that the strain of Streptomyces nojirimensis (Streptomyces nojiriensis) 9-13 had a strong antagonism against Leptospira steganensis, leptospira guotophyllum, leptospira truncatum, leptospira boningii, leptospira c.aenigma and Leptospira karst (FIG. 2).
TABLE 3 antagonism of Streptomyces nojirimensis (Streptomyces nojiriensis) Strain 9-13 against 6 Capsici fructus anthrax
Embodiment III: inhibition of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth against pepper anthracnose pathogen.
Spores of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 activated by culture medium of Gao's No. 1 are inoculated into ISP2 liquid culture medium, the fermentation temperature is 26-28 ℃, the fermentation time is 7-8d, the fermentation liquor is obtained by filtration, and then a microporous filter membrane of 0.22 mu m is sterilized for standby.
Adding 5mL of fermentation broth of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 into 50mLPDA culture medium, mixing, pouring into a culture dish, inoculating a test pathogenic bacteria cake with the diameter of 5mm into the center of a PDA culture medium plate, respectively culturing at 28 ℃ for 5-7d, and measuring colony diameter. The inhibition of hyphal growth of 6 pathogenic bacteria by the broth was calculated using a blank PDA medium as a control (table 4). The results showed that the fermentation broth of the Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 had very strong inhibitory effects on Leptospira steganensis, leptospira griseus, leptospira crassifolium, leptospira bonisatus, leptospira c.aenigma and Leptospira karst (FIG. 3).
TABLE 4 inhibition of mycelium growth of Streptomyces nojirimensis (Streptomyces nojiriensis) Strain 9-13 fermentation broth against 6 Capsici fructus anthrax
Embodiment four: inhibition of spore germination of pepper anthracnose pathogen by Streptomyces nojirimis (Streptomyces nojiriensis) strain 9-13 fermentation broth.
Inoculating pathogenic bacteria cake with diameter of 5mm into PDB liquid culture medium, shake culturing at 28deg.C and 180rpm for 7d, filtering to collect spore suspension, and adjusting its concentration to 1×10 5 And each mL.
10. Mu.L of the fermentation broth of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 in example III was added to 100. Mu.L of the spore suspension, and spore suspensions of Bacillus lazurich, leptospira fruit, anthrax grunnica, botrytis cinerea, C.aenigma and Achillea anthracis were cultured at 28℃for 7 hours, 23 hours, 3 hours, 8 hours, 5 hours and 9 hours, respectively, using the blank spore suspension as a control, and the inhibition ratio of the fermentation broth of Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 to spore germination of 6 pathogenic bacteria of pepper anthracis calculated (Table 5). The results showed that the fermentation broth of the strain 9-13 of Streptomyces nojirimensis (Streptomyces nojiriensis) significantly inhibited spore germination of Leptosporium fruit, leptosporium crenatans, leptosporum bonisatus, leptosporum C.aenigma and Leptosporum karst (FIG. 4).
TABLE 5 inhibition of germination of 6 anthrax conidia by Streptomyces nojirimensis (Streptomyces nojiriensis) Strain 9-13 fermentation broth
Fifth embodiment: the application of the fermentation liquor of the Streptomyces nojirimis (Streptomyces nojiriensis) strain 9-13 in preventing and controlling pepper anthracnose.
Culturing test of Sphaeromyces lanuginosus, botrytis cinerea, C.aenigma and karst anthracis at 28deg.C for 5d, punching bacterial cake at colony edge with punch with diameter of 5mm, transferring bacterial cake into PDB liquid culture medium, culturing at 150r/min and 28deg.C in dark shaking for 7d, detecting conidium concentration with blood cell counter, and making into 1×10 4 Mixed spore suspension of individual spores/mL for use.
The test site is selected from the Xuan Danggun in Jinan district of Fujian province, and 3 treatments are set for the test, which are respectively: a.10% difenoconazole 60 g/mu, B.fermentation broth of strain 9-13, C.clear water control. Every treatment is performed on 6 pepper plants, 4 times of repetition are performed, total 12 cells are arranged in a random group, and the spraying is performed according to the water consumption of 45 liters per mu. Spraying and applying the medicines respectively in the fruiting period of the chilli, inoculating the prepared mixed spore suspension (adding 0.1% Tween 80) after spraying and applying the medicines for 24 hours, and spraying 50mL of mixed spore suspension on each plant. The onset of disease was investigated 14-21d after inoculation.
The investigation method comprises the following steps: every treatment investigation of all pepper fruits, the total number and the number of the disease are recorded, and the disease index is counted according to the following grading standard:
level 0: the pepper fruit has no disease spots;
stage 1: the area of the disease spots accounts for 1-2% of the whole fruit area;
3 stages: the area of the disease spots accounts for 2% -5% of the whole fruit area;
5 stages: 5% -15% of the fruit areas show necrotic lesions, or up to 25% of the fruit surface water-stain lesions;
7 stages: 15% -25% of the fruit area shows necrotic lesions;
stage 9: the necrosis area accounts for more than 25% of the whole area;
disease index = Σ (number of disease fruits at each stage×number of disease grades relative to number of disease grades)/(total number of fruits investigated×9) ×100;
prevention effect (%) = [ (CK disease index-treatment disease index)/(CK disease index ] ×100;
surface 6 Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 fermentation broth field efficacy test for preventing and controlling pepper anthracnose
Experimental results show that the fermentation liquor of 10% difenoconazole WG 60 g/mu and Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 has control effects on pepper anthracnose of 75.62% and 71.85%, respectively, and is safe to peppers without causing phytotoxicity. The control effect of the fermentation liquor of the streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 on pepper anthracnose is not obvious from the control effect of 10% difenoconazole WG 60 g/mu on the 5% obvious level and 1% extremely obvious level. Therefore, the fermentation liquor of the streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13 has the control effect on pepper anthracnose equivalent to the control effect of 60 g/mu of 10% difenoconazole WG, and has good application prospect.
Claims (5)
1. The application of the Streptomyces nojirimensis strain in preventing and controlling pepper anthracnose is characterized in that: the strain is Streptomyces nojirimensis (Streptomyces nojiriensis) strain 9-13, and is preserved in China general microbiological culture Collection center (CGMCC) No.22798, and the preservation date is: 2021, 6, 28.
2. The use according to claim 1, characterized in that: the strain of Streptomyces nojirimensis (Streptomyces nojiriensis) 9-13 has antagonistic effects on Sgavill anthrax (Colletotrichum scovillei), cephalosporium carpum (C.fructicola), cephalosporium acremonium (C.truncatum), botrytis cinerea (C.boninense), zymobacter karst (C.karst ti) and C.aenigma causing pepper anthracnose.
3. The application of the fermentation liquor of the strain of the streptomyces nojirimis in preventing and controlling pepper anthracnose is characterized in that the fermentation liquor is prepared by inoculating the strain 9-13 of the strain of the streptomyces nojirimis (Streptomyces nojiriensis) in ISP2 liquid medium, fermenting at 26-28 ℃ for 7-8d and filtering.
4. Use according to claim 3, wherein the fermentation broth of the strain of streptomyces nojirimi (Streptomyces nojiriensis) 9-13 is used for inhibiting the growth of hyphae of s.gaucher anthrax (Colletotrichum scovillei), d.fruit colletotrichum (c.fructicola), c.truncatum, boningia (c.boninense), karst anthrax (c.karst ti) and c.aenigma causing pepper anthracnose.
5. Use according to claim 3, wherein the fermentation broth of the strain of streptomyces nojirimi (Streptomyces nojiriensis) 9-13 is used for inhibiting spore germination of s.gaucher anthrax (Colletotrichum scovillei), d.fruit colletotrichum (c.fructicola), c.truncatum, boningia (c.boninense), karst anthrax (c.karst ii) and c.aenigma causing pepper anthracnose.
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