CN114958613A - Trichoderma spirillum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus - Google Patents
Trichoderma spirillum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus Download PDFInfo
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- CN114958613A CN114958613A CN202210344481.2A CN202210344481A CN114958613A CN 114958613 A CN114958613 A CN 114958613A CN 202210344481 A CN202210344481 A CN 202210344481A CN 114958613 A CN114958613 A CN 114958613A
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- trichoderma
- spirillum
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- brown rot
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Images
Classifications
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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
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/12—Powders or granules
- A01N25/14—Powders or granules wettable
-
- 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
-
- 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
- C12N1/14—Fungi; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Agronomy & Crop Science (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses trichoderma spiralosum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus, wherein the trichoderma spiralosum RS05 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of institute of China academy of sciences at 12 months and 20 days in 2021, and the preservation number is CGMCC No. 40012. The Trichoderma spirillum RS05 strain and the highly-sporogenous wettable powder prepared by the strain have special effect on the brown rot of the Ma bamboo, have the inhibition rate on the pathogen of the brown rot of the Ma bamboo (Diaportheguangxiensis) of more than 70 percent, have strong environmental suitability and good affinity, are suitable for commercial production, and have wide market prospect.
Description
Technical Field
The invention relates to trichoderma spiralosum, and particularly relates to trichoderma spiralosum RS05 and application thereof in preventing and treating brown rot of dendrocalamus latiflorus.
Background
Dendrocalamus latiflorus (Dendrocalamus latiflorus) is a bamboo shoot and wood dual-purpose sympodial bamboo of Du bamboo of Bambusoideae of Gramineae, and is also an excellent bamboo plant for ecological greening. The bamboo species plays an important role in maintaining ecological balance, preventing water and soil loss, conserving water sources and the like, and has potential in aspects of biological energy, biological diversity protection, degraded land recovery and the like.
The brown rot of the Ma bamboo caused by Diaporthe guangxiensis (GenBank accession numbers: MW380383, MW431318, MW431317 and MW431316) is found in the Ma bamboo distribution region in Yangtze river basin to different degrees. The diseased bamboos firstly appear light yellow brown diseased spots at the base parts of the bamboos, the diseased spots rapidly expand upwards to form stripe spots, the color gradually deepens, and finally the diseased bamboos become purple brown to black brown, the inner wall and the node of a visible cavity of the sectioned diseased bamboos turn black, white mycelia grow, and black stripes exist in the bamboo flesh. Meanwhile, the disease spots at the basal part of the culm are also transversely expanded, and the diseased bamboos die after the disease spots surround the basal part of the culm.
The inhibition of the survival and activity of certain pathogens by inter-and intra-species competition, parasitism, bacteriolysis, etc. of microorganisms has gradually become a major technical approach for the control of plant diseases. When endophytes enter plants as disease-resistant substances and the like, the endophytes can preferentially occupy the invasion sites of pathogenic bacteria, compete with the pathogenic bacteria for nutrient substances, secrete antibacterial substances at the invasion sites and prevent the invasion of the pathogenic bacteria. Therefore, it would be of great interest to find a biocontrol method that is effective in preventing and/or treating brown rot in hemp bamboo.
Disclosure of Invention
The invention aims to provide trichoderma spiralii RS05 and application thereof in prevention and treatment of brown rot of dendrocalamus latiflorus, and solves the problem of inhibition of brown rot of dendrocalamus spiralii, and trichoderma spiralii RS05 can effectively inhibit brown rot of dendrocalamus spiralii.
In order to achieve the aim, the invention provides trichoderma spiralense RS05, wherein the trichoderma spiralense RS05 is preserved in China general microbiological culture Collection center of China institute of microbiology, academy of sciences, China, 20 months in 2021, the preservation address is No. 3 Silu 1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 40012.
The invention also aims to provide the culture medium of the Trichoderma spirillum RS05, wherein the culture medium adopts PSA culture medium as a basal culture medium; the basic culture medium contains 2% of soluble starch, 0.1-1% of tryptone and 0.01-0.1% of magnesium sulfate; or the basic culture medium contains 1% of soluble starch, 0.5% of tryptone and 0.01% of magnesium sulfate, wherein the percentage is the mass percentage of the basic culture medium.
Preferably, the basal medium contains 2% soluble starch, 0.1% tryptone and 0.05% magnesium sulfate.
Another object of the present invention is to provide a culture method for Trichoderma spirillum RS05, which comprises: culturing the Trichoderma spirillum RS05 of claim 1 in the culture medium under the culture conditions of pH 5-9, temperature 25-35 ℃, speed 100-300 r/min and ventilation of 70-130L/h, wherein the inoculation amount is 2-12%, and fermenting for 12-156 h.
Preferably, the inoculum size is 6%; the fermentation time is 96 h.
The invention also aims to provide application of the trichoderma spiralense RS05 in preventing and treating brown rot of dendrocalamus.
The invention also aims to provide Trichoderma spiralatum wettable powder for preventing and treating brown rot of Ma bamboo, which contains Trichoderma spiralatum RS 05.
Preferably, the wettable powder also contains a carrier, a wetting agent, a dispersing agent and a protective agent.
Preferably, the carrier is selected from one or more of kaolin, diatomite, calcium carbonate, talcum powder and bentonite; the wetting agent is selected from tween 20; the dispersing agent is selected from sodium lignosulfonate or/and polyethylene glycol; the protective agent is selected from humic acid or/and kaolin.
Preferably, the wettable powder is obtained by drying after mixing the concentrated fermentation liquor of the trichoderma spirillum RS05 with a carrier, a wetting agent, a dispersing agent and a protective agent; wherein the weight percentages of the carrier, the wetting agent, the dispersing agent and the protective agent and the volume percentage of the concentrated fermentation liquor of the trichoderma spiralosum RS05 are respectively 35%, 4-5%, 8-10% and 2-2.5%; or, the weight of the carrier, the wetting agent, the dispersing agent and the protective agent and the volume percentage of the concentrated fermentation liquor of the trichoderma spiraeum RS05 are respectively 40%, 4%, 10% and 1.5%.
Preferably, the spore content of the concentrated fermentation liquor of the trichoderma spirillum RS05 is 1 x 10 12 cfu/mL。
The trichoderma spiralosum RS05 and the application thereof in preventing and treating the brown rot of the dendrocalamus latiflorus solve the problem of inhibiting the brown rot of the dendrocalamus spiralosum and have the following advantages:
the Trichoderma spirillum RS05 strain and the highly-sporogenous wettable powder prepared by the strain have special effect on the brown rot of the Ma bamboo, have the inhibition rate on the pathogen of the brown rot of the Ma bamboo (Diaportheguangxiensis) of more than 70 percent, have strong environmental suitability and good affinity, are suitable for commercial production, and have wide market prospect.
Drawings
FIG. 1 shows the plate inhibition effect of the strain RS05 of the present invention on pathogenic bacteria Diaportheguangxiensis.
FIG. 2 shows the microstatic effect of the strain RS05 of the present invention on pathogenic bacteria Diaportheguangxiensis
FIG. 3 is a plate colony map of the strain RS05 of the present invention.
FIG. 4 is a microscopic morphology of the strain RS05 of the present invention.
FIG. 5 is an electrophoretogram of the strain RS05 of the present invention.
FIG. 6 is a multigene association tree based on Phyllosuite on strain RS 05.
FIG. 7 shows the effect of inoculum size and fermentation time on spore growth and bacteriostatic rate.
FIG. 8 shows the effect of different vectors on the content of Trichoderma helicatum RS 05.
FIG. 9 is a graph of the effect of different wetting agents on Trichoderma spiraeum RS05 content.
FIG. 10 is a graph of the effect of different dispersants on Trichoderma spiratum RS05 content.
FIG. 11 is a graph of the effect of different protective agents on Trichoderma helicidum RS05 content.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Experimental example 1 isolation and plate inhibition effects of biocontrol fungi
1. Isolation of biocontrol fungi
Collecting healthy dendrocalamus latiflorus rhizosphere soil in a Garcinia mangostana cultivation area in Sichuan of 6.1.2020, collecting a root system with soil layer depth of 0-20 cm in a range of 50cm around a bamboo stalk as a center, lightly shaking off soil at the periphery of the root system, and lightly brushing the soil adhered to the surface of the root with a brush to serve as rhizosphere soil. Placing 10g rhizosphere soil into a 250mL conical flask filled with 90mL sterile water, shaking uniformly to prepare 10g rhizosphere soil -1 Diluting the soil sample solution respectively 10 times -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 From the above dilutions, 100. mu.L of sterilized triangular rod was pipetted on glucose agar medium (PDA culture)The nutrient medium comprises 200g of potatoes, 20g of anhydrous glucose, 15-20 g of agar powder and 1000mL of distilled water, is sterilized at 121 ℃ for 30min under high pressure, and is uniformly coated on a streptomycin sulfate (Solarbio)) plate with the concentration of 50 mu g/mL. Each treatment is repeated for 3 times, the culture is carried out for 5-7 d at 25 ℃, single colonies with obvious colony morphology difference are picked and enter a primary screen, and 12 fungi (with the numbers of RS01, RS02, RS12) are obtained in total.
According to the differences of the colony in size, shape, color, edge characteristic and other aspects, selecting a single colony to purify on a PDA plate, and transferring the purified colony to a PDA inclined plane to store at 4 ℃ for later use.
2. Plate inhibition effect
According to the face-to-face culture method, a sterilization inoculation shovel is used for placing the pathogenic bacteria cake of the brown rot of the dendrocalamus latiflorus with the diameter of 5mm on one side of a PDA culture medium, pure microorganisms obtained by single separation are inoculated at a position 3cm away from the pathogenic bacteria cake, the culture is carried out for 7 days at the constant temperature of 25 ℃, and 3 treatments are carried out each time.
Table 1 shows the inhibition rate of each selected strain against brown rot pathogen of Ma bamboo (Diaportheguangxiensis)
Note: inhibition (%) - (treated pathogen diameter-control pathogen diameter)/control pathogen diameter × 100; data are mean ± sem, with different lower case letters after data indicating significant differences (P < 0.05).
According to the inhibition rate (see table 1) and the heavy parasitic effect of each selected bacterium on pathogenic bacteria, the strain with the best effect is selected to be RS05 (see fig. 1 and 2). As shown in FIG. 1, the plate inhibition effect of the strain RS05 on the pathogenic bacterium Diaportheguangxiensis (7 days, A: pathogenic bacterium; B: RS05) is shown, and as shown in FIG. 2, the plate inhibition effect of the strain RS05 on the pathogenic bacterium Diaportheguangxiensis (7 days, about: the entwining of the hypha of the pathogenic bacterium RS05 is the parasitism, and about: the hypha of the pathogenic bacterium is broken and swollen).
3. Identification of biocontrol fungi
(1) Morphological identification
As shown in FIG. 3, it is a plate colony map of the strain RS05 of the present invention (cultured for 5 days by PDA), the plate colony of the strain RS05 is green, the aerial hyphae are developed, and the plate colony is cultured at a constant temperature of 25 ℃ for 5 days, i.e., the plate colony is covered with green powder, and is a large number of produced conidia (FIG. 3). As shown in FIG. 4, which is a microscopic morphological diagram (5 days) of the strain RS05 of the present invention, the base morphology and the conidiophore morphology of RS05 were microscopically observed, wherein A is the short fertile lateral branch and barrel-shaped to ampoule-shaped conidiophore at the base of the conidiophore, B is the smooth rectangular to narrow elliptical conidiophore, and the short fertile lateral branch and barrel-shaped to ampoule-shaped phidiophore at the base of the conidiophore are one of the typical morphological features of Trichoderma helicidum.
(2) Molecular biological identification
Extracting DNA of the strain RS05 by adopting a fungal genome DNA extraction kit, and carrying out PCR detection on a product: the ITS, RPB2, and TEF1 genes were amplified from the obtained DNA using the High Fidelity enzyme 2 × TransTaq High Fidelity PCR Supermix, using the primers shown in Table 2 below, and the reaction system and amplification procedure shown in tables 3 and 4 below, respectively.
Table 2 shows the primer sequences used for amplifying ITS, RPB2 and TEF1 genes from the obtained DNA
Table 3 shows the PCR reaction system
Table 4 shows the PCR reaction procedure
After the PCR reaction was completed, the PCR product was analyzed by electrophoresis using a 1% agarose gel at a constant voltage of 110V for 25 min. And (3) sequencing PCR products of the fungi after agarose gel electrophoresis, logging in an NCBI GenBank database for analysis and comparison to obtain homology of each gene, determining a sequence set for tree building according to a comparison result, and determining the phylogenetic position of the target fungi through multi-gene combined tree building analysis. And (3) arranging the gene sequences for establishing the tree into an Excel table, carrying out manual approval and necessary deletion on the sequences by using MEGA software, carrying out multi-gene combined tree establishment by using PhyTOSITE, and viewing and modifying the generated phylogenetic tree by using Figtree software.
As shown in FIG. 5, it is the electrophoresis chart of the strain RS05 of the present invention, in which the electrophoresis bands (two repeats of each primer) of the products obtained by amplification of 3 primers ITS, TEF and RPB, respectively, obtain DNA fragments with lengths of 620bp, 329bp and 1137 bp. Submitting each DNA sequence of the RS05 strain to a GenBank database to obtain accession numbers (OK605030, OK905444 and OK905445), carrying out BLAST analysis, selecting a fungal DNA sequence with higher homology with the accession numbers, and carrying out multi-gene combined tree building by using PhylloSeite. As shown in fig. 6, which is a multigene combined tree based on the phyllosuite strain RS05, it can be seen from fig. 6 that the RS05 strain supports 1 branch with 100% similarity of gene nucleotide sequence and higher self-expansion value with the strain Trichoderma spiralema 022, and is far from other Trichoderma, indicating that the relationship between RS05 and Trichoderma spirale is nearest.
Based on the characteristics, the strain RS05 is classified and named as Trichoderma spirale (Trichoderma spirale), and has been preserved in the China general microbiological culture Collection center of the institute of microbiology, China institute of microbiology, 20 months in 2021, with the preservation address of No. 3 Siro-Lu 1 of Beijing, sunward, and the preservation number of CGMCC No. 40012.
4. Trichoderma spirillum RS05 fermentation optimization
(1) Screening of basal Medium
The optimal basic culture medium is selected from 10 common culture media, and the optimal basic culture medium comprises the following specific steps:
selecting PDA, PSA, PMA, Czapek culture medium, Richard culture medium, Bengal red culture medium, oatmeal agar culture medium, carrot agar culture medium, corn flour agar culture medium and tomato agar culture medium, inoculating Trichoderma spirillum RS055mm mycelium block on the solid culture medium, culturing at constant temperature of 25 deg.C for 3-5 days, and measuring colony diameter and blood each dayCalculating spore yield by ball counting plate, determining optimum basic culture medium as PSA (sucrose agar culture medium containing 200g potato, 20g agar powder and 20g sucrose), culturing on the culture medium at 3 days until colony grows full, and spore yield reaches 4.3 × 10 at 5 days 9 cfu/mL (see Table 5).
TABLE 5 direct colony and spore yield of Trichoderma spiralatum RS05 on 10 media
(2) Fermentation medium composition and proportion optimization
PSA is used as a basic culture medium, 2 percent and 1 percent (accounting for the mass percent of the basic culture medium) are respectively used as the addition amounts of a carbon source, a nitrogen source and inorganic salts for optimizing the components, dextrin, sorbitol, soluble starch, lactose and sodium acetate are selected for screening the carbon source, peptone, tryptone, beef extract, urea and yeast extract powder are selected for screening the nitrogen source, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate and magnesium sulfate are selected for screening the inorganic salts, and the influence is investigated by a single-factor test. No carbon source, nitrogen source and inorganic salt are respectively set as a control group, and the treatment is repeated for 3 times. After culturing Trichoderma spirillum RS055 at constant temperature of 25 ℃, measuring colony diameter and calculating spore yield by a blood counting plate, and determining the optimal fermentation medium components. As shown in Table 6, it was revealed that the most preferable carbon source was soluble starch, the most preferable nitrogen source was tryptone, and the most preferable inorganic salt was magnesium sulfate.
TABLE 6 direct colony and spore yield (5 days) of Trichoderma spiralatum RS05 on various carbon-nitrogen sources and inorganic salt media
And (3) setting three levels by taking the carbon source, the nitrogen source and the inorganic salt preliminarily screened in the single-factor test as factors, designing an orthogonal table to perform a liquid fermentation test, and determining the influence of each factor level on the bacteriostatic rate and the spore yield of the pathogen. Orthogonal experimental design (L9(33)) is carried out on preliminarily determined carbon sources, nitrogen sources and inorganic salts to determine the proportion of each component, and as can be seen from Table 7, the bacteriostatic rate and the sporulation quantity of the formula fermentation liquor with the addition of 2% of soluble starch, 0.1% of tryptone and 0.05% of magnesium sulfate reach the hundred-billion orders of magnitude.
Table 7 shows statistical results of the fermentation medium composition ratio optimization orthogonal test
(3) Fermentation condition optimization
Based on the determined optimal fermentation medium and the determined mixture ratio, under the culture conditions of pH 7.0, 25 ℃, 200r/min and aeration (air) of 100L/h, the inoculation amounts are respectively 2%, 4%, 6%, 8%, 10% and 12%, and the fermentation broth is extracted once every 12-156 h.
As shown in FIG. 7, the optimal inoculation amount was 6% and the optimal fermentation time was 96h (FIG. 7) in order to determine the influence of the inoculation amount and fermentation time on the spore growth amount and the bacterial inhibition rate (A: inoculation amount; B: fermentation time).
Based on the optimal inoculation amount and fermentation time, an orthogonal test is arranged according to the level of 3 factors (temperature, pH, stirring speed and ventilation) of 4 factors to measure the bacteriostasis rate (L9(34)), and the influence of the level of each factor on the bacteriostasis rate and the spore yield is measured.
As can be seen from Table 8, the optimum fermentation conditions were: the temperature is 25 ℃, the pH value is 7, the rotating speed is 200r/min, and the ventilation volume is 100L/h. Under the fermentation condition, the spore yield of the Trichoderma spirillum RS05 can be doubled on the basis of an optimal fermentation medium.
Table 8 shows the statistical results of the optimization of fermentation conditions
Experimental example 2 preparation of Trichoderma spirillum RS05 high spore type wettable powder
1. Screening of components of Trichoderma spirillum RS05 high-spore wettable powder
(1) Concentrated fermentation broth
Inoculating RS05 to the optimal fermentation medium according to the inoculation amount of 6%, and performing shake culture under the optimal fermentation condition for 96h to obtain spore suspension of biocontrol bacteria.
The content of spore liquid is fixed to 1 × 10 12 cfu/mL, placing the fermented spore liquid in a refrigerator at 4 ℃, standing for 60 hours, taking out and discarding 300mL of supernatant liquid by using an injector, and reserving the remaining 200mL of concentrated sedimentation liquid for later use.
(2) Carriers and adjuvants
Mixing the carrier kaolin, diatomaceous earth, calcium carbonate, pulvis Talci or bentonite powder at a ratio of 5% (w/v) with melted optimal culture medium, sterilizing, and making into plate.
Mixing wetting agent sodium dodecyl benzene sulfonate, tween 20, nekal, fructus Gleditsiae Abnormalis powder or tea seed cake powder with melted optimal culture medium at a concentration of 250 μ g/mL, sterilizing, and making into plate.
Mixing dispersant sodium lignosulfonate, sodium tripolyphosphate, sodium carboxymethylcellulose, polyvinyl alcohol or polyethylene glycol powder with melted optimal culture medium at a concentration of 1500 μ g/mL, sterilizing, and making into plate.
Mixing humic acid, dextrin, sodium dodecyl benzene sulfonate, kaolin or ascorbic acid powder at a concentration of 50 μ g/mL with melted optimal culture medium, sterilizing, and making into plate.
(3) Influence of different carriers and auxiliary agents on content of Trichoderma spiralatum RS05
0.1mL of the RS05 concentrated fermentation broth was passed through a column 10 6 Diluted byThereafter, the coating was applied to plates containing various carriers and auxiliaries. Culturing for 4d at 25 ℃, recording the number of colonies growing in each plate, determining the optimal carrier, wetting agent, dispersing agent and protective agent, as shown in fig. 8-11, determining the influence of different carriers and auxiliary agents on the content of Trichoderma heliciformis RS05, and determining the optimal carrier, wetting agent, dispersing agent and protective agent to be kaolin, Tween, polyethylene glycol and humic acid respectively.
The final orthogonal test was performed using the primarily screened carriers and adjuvants, and the activity and suspension rate were determined separately to determine the ratio of the components (L9(34)), see table 9, where the optimum ratio is 35% kaolin, 4% polyethylene glycol, 8% tween, and 2% humic acid (weight of components and volume of concentrated fermentation broth).
TABLE 9 orthogonal test Table for optimization of Carrier and adjuvant ratios
Note: different lower case letters after the data indicate significant differences (P < 0.05).
2. Preparation of Trichoderma spirillum RS05 high-spore type wettable powder
And mixing and stirring the concentrated fermentation liquor after optimized fermentation according to the ratio of the optimized carrier and the optimized auxiliary agent. And then drying in a drying oven at the temperature of 37 ℃ until the change range of the weight of the sample is smaller, and finally fully grinding by utilizing a grinder to prepare the high spore wettable powder.
3. Quality detection method for trichoderma spirans RS05 high-spore wettable powder
The detection method of the preparation quality is carried out according to a standard method issued by Ministry of agriculture of the people's republic of China and the State administration of quality supervision, inspection and quarantine of the people's republic of China.
The detection result is shown in Table 10, and the content of live spores in the preparation is 9.4X 10 10 cfu/g, wetting time of 61.0s (< 120s), suspension rate of 84.33% (> 70%), rate of mixed bacteria of 0.08%, pH of 7.2, fineness of 95.8%, drying decrement of 1.47%, storage stability of 93.5% or more, and all indexes of the product are higher than related national standard (NY)/T 2293.1-2012)。
Table 10 shows the results of quality tests of Trichoderma spiralatum RS05 highly sporogenous wettable powder
Experimental example 3 Trichoderma spirillum RS05 high spore type wettable powder field application example
Preparing a Trichoderma spirillum RS05 high-spore wettable powder diluent: 1g of the prepared Trichoderma spirillum RS05 high-spore wettable powder is respectively diluted by sterile water by 100 times, 200 times, 400 times, 800 times, 1200 times, 1600 times, 2400 times and 3200 times to prepare diluted solutions, and the active spore content of the diluted solutions is respectively 9.4 multiplied by 10 8 cfu/mL、4.7×10 8 cfu/mL、2.35×10 8 cfu/mL、1.18×10 8 cfu/mL、 7.83×10 7 cfu/mL、5.88×10 7 cfu/mL、3.92×10 7 cfu/mL、2.94×10 7 cfu/mL。
In the hemp bamboo cultivation area, the brown rot occurrence degree is divided into 4 occurrence areas (mild I: 5-10%, moderate II: 11-30%, severe III: 31-60%, and extremely heavy IV: 60%). And (3) treating the basal part of the dendrocalamus latiflorus by using the prepared diluent in 3-4 months, uniformly spraying along the surface, spraying 100mL of the diluent per plant once every 10 days, using sterile water as a reference, repeating for 3 times, and counting the morbidity of the dendrocalamus latiflorus. Each treatment consisted of 100 strains.
And observing the disease condition after 30 days, performing disease investigation and statistics, and calculating the disease incidence, disease index and prevention and treatment effect, wherein the calculation formula is as follows:
incidence (%) × 100 (number of diseased plants/total number of plants);
the disease index [ Σ (disease-grade number of plants × representative value)/total number of plants and × representative value of the most serious grade of disease ] × 100;
the preventing and treating effect (%) is [ (control disease index increasing rate-treatment disease index increasing rate)/control disease index increasing rate ] × 100.
TABLE 11 field control of brown rot of hemp bamboo with high spore powder of Trichoderma spiralatum
Note: different lower case letters after the data indicate significant differences (P < 0.05).
As shown in table 11, the incidence rate, disease index and control effect of brown rot of dendrocalamus latiflorus under different dilution times of trichoderma spiratum high spore powder after 30 days are recorded, and the result shows that the diluent shows good control effect on the dendrocalamus latiflorus brown rot in the test of controlling the dendrocalamus spiriferus brown rot by trichoderma RS05 high spore type wettable powder. The control effect of the 100-time dilution in four types of occurrence areas exceeds 85%, the disease spreading can be controlled, the control effect is reduced along with the increase of the dilution times, and the control effect is obviously reduced when the dilution times are 2400-3200 times. In general, in a mild disease occurrence area (below 10%), 1600-2400 times of liquid is economic concentration, and the prevention effect is about 80%. In the same way, 1200-1600 times of liquid is economic concentration in a moderate disease occurrence area (11-30%), and the prevention effect is 70-74%. In a severe disease occurrence area (31-60%), 800-1200 times of liquid is economic concentration, and the prevention effect is 70-73%. In the disease polar regeneration area (more than 60 percent), the economic concentration is 400 to 800 times, and the control effect is about 70 percent. In summary, the Trichoderma spirillum RS05 strain and the high-spore wettable powder prepared by the same have special effect on the brown rot of Ma bamboo, have strong environmental suitability and good affinity, are suitable for commercial production, and have wide market prospect.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (10)
1. Trichoderma spiralatum RS05, wherein Trichoderma spiralatum RS05 has been preserved in China general microbiological culture Collection center of China institute of microbiology, academy of sciences, 20 months at 2021, with the preservation number CGMCC No.40012, No. 3 of Xilu No. 1 of Beijing north Chen of the sunward area.
2. A culture medium for Trichoderma spiralense RS05 according to claim 1, wherein the culture medium is PSA culture medium as basal medium;
the basic culture medium contains 2% of soluble starch, 0.1-1% of tryptone and 0.01-0.1% of magnesium sulfate; alternatively, the basal medium contains 1% soluble starch, 0.5% tryptone and 0.01% magnesium sulfate.
3. The culture medium of claim 2, wherein the basal medium comprises 2% soluble starch, 0.1% tryptone, and 0.05% magnesium sulfate.
4. A culture method for Trichoderma spiraling RS05 according to claim 1, comprising:
culturing Trichoderma spirillum RS05 of claim 1 in the culture medium of claim 2 or 3 at an inoculum size of 2-12% under the culture conditions of pH 5-9, temperature 25-35 ℃, air flow of 100-300 r/min and air flow of 70-130L/h, and fermenting for 12-156 h.
5. The culture method according to claim 4, wherein the inoculum size is 6%; the fermentation time is 96 h.
6. Use of trichoderma spirillum RS05 of claim 1 for the control of brown rot of dendrocalamus latiflorus.
7. A wettable powder of Trichoderma spirillum for preventing and treating brown rot of Ma bamboo, which contains Trichoderma spirillum RS05 of claim 1.
8. The wettable powder of claim 7 further comprising a carrier, a wetting agent, a dispersing agent and a protecting agent.
9. The wettable powder of claim 8 wherein the carrier is selected from any one or more of kaolin, diatomaceous earth, calcium carbonate, talc and bentonite; the wetting agent is selected from tween 20; the dispersing agent is selected from sodium lignosulfonate or/and polyethylene glycol; the protective agent is selected from humic acid or/and kaolin.
10. The wettable powder as claimed in claim 8, which is obtained by drying the concentrated fermentation broth of trichoderma spiraeanum RS05 after mixing with the carrier, wetting agent, dispersing agent and protecting agent; wherein the weight percentages of the carrier, the wetting agent, the dispersing agent and the protective agent and the volume percentage of the concentrated fermentation liquor of the trichoderma spiralosum RS05 are respectively 35%, 4-5%, 8-10% and 2-2.5%; or, the weight of the carrier, the wetting agent, the dispersing agent and the protective agent and the volume percentage of the concentrated fermentation liquor of the trichoderma spiraeum RS05 are respectively 40%, 4%, 10% and 1.5%.
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| CN116267998B (en) * | 2023-01-04 | 2024-06-04 | 四川农业大学 | Compound preparation for resisting diseases and promoting growth and application thereof |
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