CN117327593A - Trichoderma harzianum ZM1 and application thereof - Google Patents
Trichoderma harzianum ZM1 and application thereof Download PDFInfo
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- 235000020232 peanut Nutrition 0.000 claims abstract description 61
- 235000017060 Arachis glabrata Nutrition 0.000 claims abstract description 51
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- PZBPHYLKIMOZPR-FIYGWYQWSA-K 2-[4-[2-[[(2r)-1-[[(4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-4-[[(2r,3r)-1,3-dihydroxybutan-2-yl]carbamoyl]-7-[(1r)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicos-19-yl] Chemical compound [68Ga+3].C([C@H](C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)NC(=O)CN1CCN(CC([O-])=O)CCN(CC([O-])=O)CCN(CC([O-])=O)CC1)C1=CC=CC=C1 PZBPHYLKIMOZPR-FIYGWYQWSA-K 0.000 claims description 4
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- HZRSNVGNWUDEFX-UHFFFAOYSA-N pyraclostrobin Chemical compound COC(=O)N(OC)C1=CC=CC=C1COC1=NN(C=2C=CC(Cl)=CC=2)C=C1 HZRSNVGNWUDEFX-UHFFFAOYSA-N 0.000 description 7
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- 241001503951 Phoma Species 0.000 description 2
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- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 1
- 241000221662 Sclerotinia Species 0.000 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to trichoderma harzianum ZM1 and application thereof, which are preserved in the China general microbiological culture collection center (CGMCC) with the preservation number of 23300. The Trichoderma harzianum ZM1 provided by the invention is a biocontrol strain, and can inhibit peanut pathogenic bacteria, prevent and treat peanut southern blight and promote peanut growth when applied to the prevention and treatment of peanut pathogenic bacteria. The strain ZM1 improves the resistance of peanut aligned sclerotium rolfsii, sclerotinia sclerotiorum, botrytis cinerea, pythium gracile, net blotch germ and leaf rot germ, has obvious effect, is safe and quick, is environment-friendly, is beneficial to the green and healthy production of peanuts, and provides technical support for the comprehensive control of peanut diseases.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to a biocontrol fungus Trichoderma harzianum, and in particular relates to separation and identification of Trichoderma harzianum ZM1 with antibacterial activity on various peanut pathogenic fungi and application of the trichoderma harzianum ZM1 in promoting peanut growth and preventing and controlling peanut pathogenic fungi.
Background
The peanuts are damaged by various pathogenic microorganisms in the whole growth period, and diseases seriously threaten the peanut production, so that the peanut yield and quality are reduced. The health production of peanuts is severely affected by sclerotium disease caused by sclerotium rolfsii (Sclerotium rolfsii), sclerotium bacteria caused by sclerotium rolfsii (Sclerotinia sclerotiorum), gray mold caused by Botrytis cinerea (Botrytis cinerea), rot caused by Pythium gracile (Pythium myriotylum), leaf rot caused by rhizoctonia solani (Rhizoctonia solani), net blotch caused by phoma arachidosum (Phoma arachidicola) and the like.
In peanut production, bactericides are currently relied largely on to control diseases, but they have a number of adverse consequences for human, animal health and environmental safety; therefore, biological control is particularly important in peanut disease control.
Among microorganisms, trichoderma is currently the most effective biocontrol factor used because of its ability to produce various antibacterial metabolites, its wide adaptability to temperature, humidity and pH, its fast propagation speed and its strong saprophytic property. Trichoderma is a ubiquitous and easily-separated filamentous fungus, is commonly found in soil, and in biocontrol preparations for preventing and controlling plant pathogenic fungi, the trichoderma biocontrol preparations account for a large proportion, and account for more than 60% in 2015 according to statistics. In the commercialized trichoderma biological preparation, the most studied and applied trichoderma species are trichoderma harzianum, and the trichoderma harzianum is parasitic on plant roots for a long time, so that the improvement of the tolerance of plants to abiotic stress and biotic stress is facilitated, and the growth of plants is promoted. At present, in the control method of peanut pathogenic bacteria, the trichoderma strains and related application products are fewer, so that the trichoderma harzianum and the application technology thereof for efficiently antagonizing the peanut pathogenic bacteria are very necessary and urgent to screen and develop based on the background of sustainable development of agriculture and ecological environment protection.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides trichoderma harzianum ZM1 and application thereof in view of the advantages of biological control in peanut disease control, the trichoderma harzianum ZM1 is applied to peanuts, and various peanut pathogenic bacteria are controlled by means of a biocontrol strain, so that the effect is obvious and the method is environment-friendly, and meanwhile, the strain can promote the growth of the peanuts.
The technical scheme of the invention is as follows:
trichoderma harzianum (Trichoderma harzianum) ZM1 is preserved in China general microbiological culture Collection center with a preservation number of CGMCC No.23300.
The invention also provides application of the Trichoderma harzianum ZM1 in inhibiting peanut pathogenic bacteria, wherein the peanut pathogenic bacteria are any one or more of sclerotium rolfsii, sclerotinia sclerotiorum, gray mold bacteria, pythium gracile, net spot bacteria or leaf rot bacteria.
Further, the application form is a cake or spore suspension of the strain ZM1.
The invention also provides application of the Trichoderma harzianum ZM1 in preventing and treating peanut southern blight; and application of trichoderma harzianum ZM1 in promoting peanut growth.
Further, the application form is spore suspension of strain ZM1.
The invention also provides a microbial inoculum for preventing and controlling peanut pathogenic bacteria, wherein the active ingredient of the microbial inoculum is trichoderma harzianum ZM1, and the peanut pathogenic bacteria are any one or more of sclerotium rolfsii, sclerotinia sclerotiorum, botrytis cinerea, pythium group, net spot bacteria and leaf rot bacteria.
Specifically, the trichoderma harzianum ZM1 provided by the invention is used for preventing and treating sclerotium rolfsii caused by sclerotium rolfsii, gray mold caused by botrytis cinerea, rotten fruit disease caused by Pythium group, leaf rot caused by rhizoctonia solani and net blotch caused by phoma of peanut.
Biological material sample preservation information:
trichoderma harzianum (Trichoderma harzianum) ZM1, deposited in China general microbiological culture Collection center (CGMCC), deposit address: the national institute of microbiology, national academy of sciences, china, collection date: 2021, 11 and 05 days, and the preservation number is CGMCC No.23300.
The invention has the beneficial effects that:
the Trichoderma harzianum ZM1 provided by the invention is used for preventing and controlling peanut pathogenic bacteria, so that the resistance of the peanut aligned sclerotium rolfsii, sclerotinia sclerotiorum, pythium gracile, and brown spot bacteria and leaf rot bacteria is improved, the effect is obvious, and the growth of the peanut can be promoted; the method belongs to the category of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, and provides technical support for comprehensive control of peanut diseases.
Drawings
FIG. 1 is a morphological feature of Trichoderma harzianum ZM1 strain;
FIG. 2 is a phylogenetic tree of Trichoderma harzianum ZM1 constructed based on tef1 and rpb gene fragments;
FIG. 3 is a schematic diagram showing the antagonism of Trichoderma harzianum ZM1 against 6 pathogens; wherein, fig. 3A is a graph showing antagonism effect of ZM1 against 6 peanut pathogens; FIG. 3B is a graph showing colony growth statistics of 6 peanut pathogens; FIG. 3C is a graph showing statistics of the bacteriostatic rate of ZM1 against 6 peanut pathogens; * Or capital letters indicate that the difference is very significant at the 0.01 level.
Detailed Description
For a further understanding of the present invention, reference will now be made in detail to the present embodiments of the invention, examples of which are illustrated in the accompanying drawings and described in a complete and clear manner. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Separation and identification of Trichoderma harzianum ZM1
(1) Isolation of Strain ZM 1: about 100g of rhizosphere soil 5-10 cm away from the ground surface is collected from the land where peanut southern blight in Laiyi is serious, and the rhizosphere soil is brought back to a laboratory and stored at 4 ℃. Separating trichoderma by adopting a dilution coating flat plate method: adding 10g of soil sample into 90mL of sterile water, oscillating at 25 ℃ and 140rpm for 10min, standing for 5min, collecting supernatant, and sequentially diluting to 10 times by adopting a 10-time serial dilution method -2 、10 -3 、10 -4 And 10 -5 100. Mu.L of each concentration was pipetted onto PDA medium and each concentration was repeated 3 times and incubated at 25℃in dark. Spraying the spore suspension of the peanut crown rot germ when fungus colonies appear on the culture medium. After 3d of culture, selecting colonies with obvious bacteriostasis areas around, picking single colonies by using a bacteria transferring ring, and purifying and culturing on a PDA culture medium. A strain with obvious inhibition zone and green thallus is selected and named ZM1 as shown in figure 1. Transferring the purified ZM1 strain to a PDA inclined plane for standby.
(2) Identification of Strain ZM 1: the spores and mycelia of ZM1 were collected, and the genome was extracted and used as a template for PCR amplification using the primers EF1-728F (5'CATCGAGAAGTTCGAGAAGG 3') (SEQ ID NO: 3), EF1-986R (5'TACTTGAAGGAACCCTTACC 3') (SEQ ID NO: 4) and RPB2 gene primers RPB2-5F (5'GAYGAYMGWGATCAYTTYGG 3') (SEQ ID NO: 5), RPB2-7cR (5'CCCATRGCTTGYTTRCCCAT 3') (SEQ ID NO: 6) of the tef1 gene, respectively. The PCR products were sequenced by Beijing engine biotechnology Co. the total length of the tef1 gene sequence is 330bp, and the sequence is shown in SEQ ID NO:1 is shown in the specification; the total length of the rpb gene sequence is 1044bp, and the sequence is shown in SEQ ID NO: 2.
BLAST analysis and comparison are carried out on the obtained sequence, and a phylogenetic tree is constructed by combining tef1 gene and rpb gene segments through MEGA7 software, as shown in figure 2, and the result shows that the strain ZM1 and Trichoderma harzianum Trichoderma afroharzianum are on the same branch, and the strain ZM1 is Trichoderma harzianum. The strain ZM1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23300.
Example 2
Inhibition effect of bacterial strain ZM1 bacterial cake on growth of 6 peanut pathogenic bacteria hyphae
Respectively inoculating ZM1, rhizoctonia solani, sclerotinia sclerotiorum, botrytis cinerea, pythium gracile, leptosphaeria gracilis and leaf rot fungi to the center of a PDA culture medium, and culturing in dark at 25 ℃ for 7d for later use. The PDA culture medium is inoculated with a bacterial cake of pathogenic bacteria with the diameter of 5mm at the center, ZM1 bacterial cakes with the diameter of 5mm are placed at the positions of about 20mm on two sides of the bacterial cake, ZM1 bacterial cakes are not inoculated as a control, each treatment is repeated 6 times, and the culture is carried out at the constant temperature of 25 ℃.
As shown in fig. 3A, the antagonism effect of ZM1 against 6 peanut pathogens; measuring the growth condition of hypha, and obtaining the growth diameter of the bacterial colony from the ordinate, wherein the bacterial colony is the bacterial colony growth statistical result of 6 peanut pathogenic bacteria as shown in FIG. 3B; calculating the bacteriostasis rate by adopting a hypha growth rate method: antibacterial ratio (%) = (control colony growth diameter-treated colony growth diameter)/control colony growth diameter×100. As a result, it was found that the strain ZM1 had a remarkable bacteriostatic effect against 6 pathogenic bacteria (see FIG. 3). As shown in FIG. 3C, the mycelial growth inhibition rates of ZM 1-aligned sclerotinia, sclerotinia sclerotiorum, botrytis cinerea, pythium gracile and Pythium gracile were 78.83%, 74.21%, 86.71%, 82.60%, 86.47% and 86.98%, respectively.
Example 3
Effect of Strain ZM1 spore suspension on potted peanut growth and southern blight resistance
Sterilizing peanut seed and flower 33, sowing in flowerpot, culturing in greenhouse with good illumination at 25deg.C, and culturing in strain ZM1 spore suspension (1×10 after 2 weeks 6 spores/mL, 5 mL/strain) were rooted and inoculated with an equal volume of PDB broth as control. 20 pots of control group (CK) and treatment group (ZM 1) were each repeated 3 times, 10 pots of peanuts of treatment group were taken after 4 weeks each, the total fresh mass of the plants was weighed, and the total dry mass of the plants was weighed after drying. And inoculating oat grains full of sclerotium rolfsii mycelia on the base of the rest peanut stems, investigating the occurrence condition of southern blight after 6 weeks, and calculating the control effect.
Classification standard of disease index of peanut southern blight in seedling stage: level 0: no disease spots; stage 1: only the stems have disease spots, and the root systems are healthy; 2 stages: the stem and root system generate disease spots, and the overground part is normal; 3 stages: the overground parts show wilting symptoms, which are mainly manifested by side branch wilting or withering; 4 stages: symptoms of wilting or dying of the main stem appear. Disease index = Σ (value of each level×number of disease plants of each level)/(highest level×total number of investigation) ×100, incidence (%) = number of disease plants/total number of investigation×100%, control effect (%) = (control disease index-treatment disease index)/control disease index×100%.
The statistical results are shown in the following table 1, and compared with a control group, the bacterial strain ZM1 can remarkably promote the growth of flower-growing No. 33 and improve the resistance to southern blight; compared with a control group, the strain ZM1 spore suspension can remarkably increase the fresh quality and the dry quality of a flower 33 plant by 27.45 percent and 24.57 percent respectively; mortality from southern blight was reduced by 79.59% in the treated group compared to the control group; the effect of strain ZM1 on preventing and treating southern blight was 56.80% (Table 1).
TABLE 1 control Effect of ZM1 on peanut growth and southern blight in greenhouse
| Fresh quality (g) | Dry mass (g) | Mortality (%) | Disease condition refers to the condition of a patientNumber (%) | Preventing effect (%) | |
| CK | 10.51±0.79A | 1.45±0.13A | 68.06±6.36A | 84.38±2.76A | / |
| ZM1 | 14.49±1.54B | 1.92±0.20B | 13.89±2.41B | 36.16±1.04B | 56.80±1.23 |
Example 4
Effect of Strain ZM1 spore suspension on southern blight resistance of peanut in field
3 treatments were applied to the plot where peanut southern blight occurred severely, ZM1 spore suspension (1X 10 6 spore/mL) root irrigation treatment (ZM 1), 30% pyraclostrobin 2000-fold aqueous root irrigation treatment (pyraclostrobin), and clear water Control (CK). 10m per cell using random block design 2 Repeated 4 times. Sowing flower 33 in middle ten days of 5 months, and at the beginning of 7 months, respectively irrigating roots with ZM1 spore suspension, 30% pyraclostrobin 2000 times aqueous solution and clear water (10 mL/plant), irrigating roots once every 2 weeks, irrigating roots 2 times altogether, and investigating the disease condition of southern blight when harvesting peanuts in middle ten days of 9 months.
Grading standard of disease index of peanut southern blight in harvest period: level 0: the plant has no disease spots; stage 1: only the aerial parts or root systems have lesions and the plants are not dead; 2 stages: the plant is not dead, the pod spot is less than or equal to 10 percent, or the plant is dead, and the pod has no spot; 3 stages: the plant is not dead, the pod spot is less than or equal to 30 percent, or the plant is dead, and the pod spot is less than or equal to 10 percent; 4 stages: the plants did not die, the pod spotting was > 30%, or the plants die, the pod spotting was > 10%, or there was no yield. Disease index = Σ (value of each level×number of disease plants of each level)/(highest level×total number of investigation) ×100, incidence (%) = number of disease plants/total number of investigation×100%, control effect (%) = (control disease index-treatment disease index)/control disease index×100%.
The statistical results are shown in the following table 2, and compared with a control, the bacterial strain ZM1 and pyraclostrobin can remarkably improve the resistance of peanuts to southern blight. After the clear water, the pyraclostrobin and the ZM1 are treated, the death rate of the peanuts is 83.33%, 10.83% and 12.50% respectively; the disease indexes are 94.79%, 41.67% and 44.38% respectively; the control effects of pyraclostrobin and ZM1 on southern blight are 56.24% and 53.39% respectively, and the difference between the two is not obvious (Table 2).
TABLE 2 prevention and treatment effects of ZM1 in field on peanut southern blight
| Treatment of | Mortality (%) | Index of disease (%) | Preventing effect (%) |
| CK | 83.33±2.72A | 94.79±2.19A | / |
| Pyraclostrobin | 10.83±3.19B | 41.67±3.04B | 56.24±3.20A |
| ZM1 | 12.50±1.67B | 44.38±2.29B | 53.39±2.41A |
The foregoing description is only a preferred embodiment of the present invention and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any modification, equivalent replacement, variation, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. Trichoderma harzianum (Trichoderma harzianum) ZM1 is preserved in China general microbiological culture Collection center with a preservation number of CGMCC No.23300.
2. The application of trichoderma harzianum ZM1 as claimed in claim 1 in inhibiting peanut pathogenic bacteria, wherein the peanut pathogenic bacteria are any one or more of sclerotium rolfsii, sclerotinia sclerotiorum, botrytis cinerea, pythium group, net spot bacteria or leaf rot bacteria.
3. Use according to claim 2, characterized in that the form of application is a cake or spore suspension of strain ZM1.
4. The use of trichoderma harzianum ZM1 as claimed in claim 1 for controlling peanut southern blight.
5. Use of trichoderma harzianum ZM1 as described in claim 1 for promoting peanut growth.
6. The use according to any one of claims 4 or 5, characterized in that the form of use is a spore suspension of strain ZM1.
7. The microbial inoculum for preventing and controlling peanut pathogenic bacteria is characterized in that the active ingredient of the microbial inoculum is Trichoderma harzianum ZM1 in claim 1, and the peanut pathogenic bacteria are any one or more of sclerotium rolfsii, sclerotinia sclerotiorum, botrytis cinerea, pythium group, net spot bacteria or leaf rot bacteria.
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| CN202311568179.6A CN117327593A (en) | 2023-11-23 | 2023-11-23 | Trichoderma harzianum ZM1 and application thereof |
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| CN202311568179.6A CN117327593A (en) | 2023-11-23 | 2023-11-23 | Trichoderma harzianum ZM1 and application thereof |
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