CN114874917B

CN114874917B - Trichoderma atroviride T3, microbial inoculum prepared from same, microbial inoculum preparation method and application of microbial inoculum

Info

Publication number: CN114874917B
Application number: CN202210460472.XA
Authority: CN
Inventors: 尚淑梅; 刘燕; 代源源
Original assignee: Yangtze Normal University
Current assignee: Yangtze Normal University
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2023-05-23
Anticipated expiration: 2042-04-28
Also published as: CN114874917A

Abstract

The invention relates to the technical field of plant disease microorganism control, in particular to trichoderma atroviride T3, a microbial inoculum prepared from the trichoderma atroviride T3, a microbial inoculum preparation method and application of the microbial inoculum. The preservation number of the trichoderma atroviride (Trichoderma atroviride) T3 is CCTCC NO: M2022330, the trichoderma atroviride T3 can inhibit the growth of fusarium putrescens, fusarium oxysporum and fusarium tricornutum, and the spore suspension prepared by fermenting and culturing the trichoderma atroviride can remarkably reduce the morbidity and the disease index by root irrigation treatment of coptis chinensis disease plants, improve the root rot prevention effect, and have wide application prospect.

Description

Trichoderma atroviride T3, microbial inoculum prepared from same, microbial inoculum preparation method and application of microbial inoculum

Technical Field

The invention relates to the technical field of plant disease microorganism control, in particular to trichoderma atroviride T3, a microbial inoculum prepared from the trichoderma atroviride T3, a microbial inoculum preparation method and application of the microbial inoculum.

Background

Rhizoma Coptidis (Coptis chinensis) is a perennial herb of Ranunculaceae, and has antibacterial, antioxidant, antitumor, and blood glucose lowering effects, and can be widely used in medical treatment and health promotion industry. The continuous expansion of the cultivation and planting area of coptis chinensis in successive years leads to aggravation of root rot attack, seriously influences the yield and quality of coptis chinensis, seriously reduces the enthusiasm of peasant coptis chinensis, and seriously restricts the industrialized development of coptis chinensis.

The root rot of coptis is caused by complex infection of various microorganisms, and pathogenic bacteria are complex and various, wherein fusarium strains are strong pathogenic bacteria of coptis. In production, pathogenic bacteria of the root rot of coptis infects roots or stems of coptis when meeting growth conditions of the pathogenic bacteria, but the pathogenic bacteria are not easily perceived in the early stage of the disease, the control effect in the later stage of the disease is poor, and the whole coptis is dead when serious. At present, the prevention and treatment of the coptis root rot is mainly chemical prevention and treatment, and the use of chemical preparations for sterilizing the soil not only can damage the ecological structure of the soil, but also can pollute the environment. Therefore, it is urgent to find a more efficient and pollution-free control method.

Biological pesticides are more and more favored by people because of safety, effectiveness and environmental friendliness, and are an important component of future agricultural development. At present, research on coptis root rot at home and abroad is mainly focused on disease investigation and separation and identification of pathogenic bacteria, but research on biological control of coptis root rot is still blank.

Disclosure of Invention

In order to fill the blank of the prior art, the invention provides trichoderma atroviride T3, a microbial inoculum prepared from the trichoderma atroviride T3, a microbial inoculum preparation method and application of the microbial inoculum.

In a first aspect, the invention provides Trichoderma atroviride (Trichoderma atroviride) T3 having a accession number of CCTCC NO: M2022330.

In a second aspect, the invention provides a microbial inoculum for preventing and treating root rot of coptis, which is spore suspension prepared by fermenting and culturing trichoderma atroviride T3.

Further, the pathogenic bacteria of the coptis root rot are Fusarium putrescens, fusarium oxysporum and Fusarium tricornutum.

In a third aspect, the invention provides a preparation method of the microbial inoculum for preventing and treating coptis root rot, which comprises the following steps:

taking Trichoderma atroviride T3 strain, picking a small amount of mycelium under aseptic condition, inoculating into sterilized PDA culture medium, dark culturing at 30deg.C and rotation speed of 180r/min for 5-6d, sucking clear liquid, and diluting to obtain spore suspension.

In a fourth aspect, the invention provides an application of the microbial inoculum for preventing and treating coptis root rot in preventing and treating coptis root rot, and the spore suspension is applied to root irrigation of coptis disease plants.

Further, the spore suspension has a concentration of 0.8 to 1X 10 ⁶ And each mL.

The invention has the following beneficial effects:

the trichoderma atroviride T3 can inhibit the growth of fusarium solani, fusarium oxysporum and fusarium tricuspidatum, reduce the disease index of the root rot of coptis, has a control effect of 86.36%, is nuisance-free and pollution-free, and has a wide application prospect.

Drawings

FIG. 1 is a colony characterization of Trichoderma viride T3 in different media, wherein A: PDA medium; SNA culture medium; c: CMD medium; d: EMA medium.

FIG. 2 shows the mycelium and spore morphology of Trichoderma atroviride T3, wherein A is the mycelium microscopic morphology of T3, B, C is the spore stalk of T3, and D is the spore morphology of T3.

FIG. 3 is a phylogenetic tree based on ITS-TEF combined sequences.

FIG. 4 shows the results of a plate counter experiment, wherein A is an HL-01 control, D is an HL-01 counter front, and J is an HL-01 counter back; b is an HL-05 contrast, E is an HL-05 counter front, and H is an HL-05 back; c is HL-15 contrast, F is HL-15 opposite front, and I is HL-15 opposite back.

FIG. 5 shows the effect of temperature on Trichoderma atroviride T3 sporulation.

FIG. 6 shows the effect of rotational speed on the sporulation yield of Trichoderma atroviride T3.

FIG. 7 shows the effect of inoculum size on Trichoderma atroviride T3 sporulation

FIG. 8 shows the control effect of Trichoderma atroviride T3 fermentation broth on Fusarium oxysporum of root rot of Coptis, and the control group is inoculated with pathogenic bacteria Fusarium oxysporum HL-05 only.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

Example 1: isolation and identification of strains

1. Method of

1.1 sample collection

In 2018, 3 parts of healthy coptis root soil samples, 100g each, are collected in the field (30 degrees 10 in North latitude and 108 degrees 35 in east longitude) where root rot of coptis is frequently caused in Shizhu county yellow Shui Zhen in Chongqing. Collecting rhizosphere soil of Coptidis rhizoma by root shaking method, placing into ice box, and immediately returning to laboratory for use at 4deg.C.

1.2 screening of biocontrol Trichoderma

10g of the soil sample is accurately weighed and placed in a 250mL triangular flask containing 90mL of physiological saline and glass beads. Made into 10 ^-1 Is placed in a shaking table (28 ℃ C., 150 r/min) for culturing for 30min, 1mL of supernatant is taken and subjected to gradient dilution to prepare 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 The suspensions were applied uniformly to Ma Dingshi-Bengalhond plates at a concentration of 0.1mL each, and incubated in the dark at 28 ℃. After the colony grows out, a new plate is transferred by adopting a punching inoculation method, then a single spore purification method is adopted to purify strains, single cell colonies are picked up and placed on a PDA culture medium, and after the inclined plane grows up, the strains are preserved at 4 ℃ for standby.

Screening biocontrol trichoderma by adopting a plate facing method, inoculating separated and purified trichoderma and pathogenic bacteria (the tested coptis pathogenic bacteria are fusarium putrescens (fusarium. Solani) HL-01, fusarium oxysporum (fusarium. Trichum) HL-15, (the fusarium trichum is separated and stored by modern agriculture and bioengineering academy of sciences of the Yangtze university) in a PDA culture medium by using a 6mm sterile puncher, inoculating the pathogenic bacteria in the center of the plate, inoculating the trichoderma to the position 25mm away from the center of the plate, inoculating 2 points on each dish, performing a linear shape, culturing in a PDA plate inoculated with the pathogenic bacteria only at constant temperature of 28 ℃, and measuring the colony diameter of the pathogenic bacteria when the control plate is full, so as to calculate the antibacterial rate.

Antibacterial ratio = (control pathogen plaque diameter-treatment pathogen plaque diameter)/control pathogen plaque diameter x 100%.

1.3 identification of species

1.3.1 morphological identification

The purified biocontrol trichoderma is respectively inoculated on PDA, SNA, CMD and EMA plates, placed in dark at 28 ℃ for 7d, colony morphology and color are observed, microscopic morphological characteristics of hyphae, conidiophores and conidiophores of the biocontrol trichoderma cultured on a PDA plate are observed, and morphological identification of the biocontrol trichoderma strain is carried out according to a Gams & Bissett (1998) classification system.

1.3.2 determination of the sequence of the rDNA-ITS, TEF1 Gene of biocontrol Trichoderma

Trichoderma genomic DNA extraction was performed as described by the fungal genomic DNA extraction kit (Solarbio, D2300) using the fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID No. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID No. 2) to amplify Trichoderma rDNA ITS. PCR reaction system: 1. Mu.L of DNA template, 3. Mu.L of 5 XBuffer, 2. Mu.L of dNTP, 3. Mu.L of primers, 0.2. Mu.L of enzyme, and dd H ₂ O30. Mu.L. PCR reaction conditions: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min,35 cycles; finally, the extension is carried out for 10min at 72 ℃. Trichoderma TEF1 was amplified using primers EF1-526F (5 '-GTCGTYGTYATYGGHCAYGT-3', shown in SEQ ID NO. 3) and EF1-1567R (5 '-ACHGTRCCRATAC CACCRATCTT-3', shown in SEQ ID NO. 4). PCR reaction system: 1. Mu.L of DNA template, 3. Mu.L of 5 XBuffer, 2. Mu.L of dNTP, 3. Mu.L of primers, 0.2. Mu.L of enzyme, and dd H ₂ O30. Mu.L. PCR reaction conditions: pre-denaturation at 95℃for 5min, denaturation at 95℃for 30s, annealing at 63℃for 55s, extension at 72℃for 90s,35 cycles; finally, the extension is carried out for 10min at 72 ℃. The PCR products of rDNA ITS and TEF1 were separately gel recovered (Solarbio, D2500-2) and submitted to Shanghai Biotechnology Inc. for sequencing. Sequencing results were registered in GenBank for BLAST homologous sequence alignment analysis.

1.3.3 double Gene Joint construction System evolutionary Tree

The sequences of the T3 strain rDNA ITS and TEF1 genes obtained by sequencing and the rDNA ITS and TEF1 genes of other strains of trichoderma selected from GenBank are aligned and corrected manually by MEGAX, and the corrected sequences are connected end to end according to the sequence of ITS-TEF 1. A phylogenetic tree of 22 strains including T3 strains was constructed by using the maximum likelihood method (maximum likelihood, ML) of MEGAX software, and the bootstrap support value (boottrap) was set to 1000, with Protocrea farinosa CBS121551 and Protocrea pallida CBS 299.78 as outer groups.

2. Results

2.1 isolation of species and colony morphology thereof

2 strains of fungi are separated from coptis rhizosphere soil, are respectively numbered as T2 and T3 according to colony morphology, are subjected to single spore purification and are preserved for standby (antagonism experiment results show that the T2 strain has no antagonism, and only T3 data are displayed for the purpose). As shown in FIG. 1, the fastest growth occurred in PDA, EMA, 7d hyphae overgrown the plates, 11d hyphae overgrown the plates in CMD, and 15d hyphae overgrown the plates in SNA. In PDA, aerial hyphae are dense and vigorous, the hyphae are villiated and continuously spread to the distance, the spore clusters are annular, the initial white color is gradually changed into light green and then into dark green, the spore clusters in the middle layer are denser, the inner rings of the spore clusters are wider and denser, the outer rings of the spore clusters are sparse, and the spore clusters are unevenly distributed. In SNA, hyphae are few and grow closely to the surface of the culture medium, the culture medium is sparse and not vigorous, aerial hyphae are not seen, spore clusters are annular but unevenly distributed, and the outermost spore clusters are sparse but dark green. Hypha in CMD does not grow vigorously, is not dense, is in a plush shape, has spore clusters in a ring shape, has a dark green inner layer and has a light green or slightly yellow outer layer. Hypha in the EMA is outwards diffused in a plush shape, the hypha is sparse, spore clusters are distributed in a ring shape, the outer layer is dark green, the inner layer is green, and the outer layer is slightly lighter.

2.2 microscopic morphology of the seed

The hypha is simply separated, tree branches, and the diameter of the hypha is 3.1-5.2 mu m; the conidiophore is bottle-shaped, and the conidiophore grows reciprocal branches and opposite branches, and the top end is conidiophore; conidium is light green, single cell, oval or round, and has a size of length (4.0-5.0) μm by width (3.0-4.0) μm (FIG. 2).

2.3 molecular identification of species and phylogenetic analysis

The ITS sequence length of the strain T3 is 566bp, and the TEF1 sequence length is 1273bp; the gene sequences have been uploaded to the Genbank database under accession numbers OM570594 and ON149861, respectively. Blast comparison is carried out by using ITS and TEF1 gene sequences respectively, and T3 is similar to Trichoderma strains, wherein the similarity of ITS and Trichoderma atroviride CBS 142.95 is the highest and is 99.46%; the highest similarity between TEF1 and Trichoderma atroviride IMI 206040 was 99.61%. Selecting standard strains of Trichoderma, taking 2 strains of Protocreatea as exogenons, constructing a systematic construction tree based on ITS and TEF1 gene sequence combination, and clustering T3 to Trichoderma atroviride branches. Based on morphological and systematic evolution analysis, T3 was identified as Trichoderma atroviride (FIG. 3), classified as Trichoderma atroviride (Trichoderma atroviride), and deposited at China Center for Type Culture Collection (CCTCC) for 28 days at 2022 with a deposit number of CCTCC NO: M2022330 at university of Wuhan, china.

2.4 antagonism of Trichoderma atroviride T3 on Coptis root rot

As shown in FIG. 4 and Table 1, after the counter experiment inoculation, the growth rate of Trichoderma was observed to be significantly higher than that of the pathogenic bacteria HL-01, HL-05, HL-15, wherein the HL-01, HL-05 colonies grew slightly, and the HL-15 grew slowest and contacted the pathogenic bacteria after 5d,8d and 7d, respectively, after which the pathogenic bacteria stopped growing forward. At the opposite junction of HL-01 and HL-05, a bacteriostatic zone is formed to generate yellow brown pigment, and the yellow brown pigment on the back of the flat plate is more obvious; and no pigment is produced at the opposite junction of the front and back surfaces of HL-15. The trichoderma has the highest antibacterial rate on fusarium tricuspension HL-15, the antibacterial rate is up to 85.98 percent, and the antibacterial rate is 77.71 percent after fusarium oxysporum HL-05; furthermore, fusarium solani HL-01 has a bacteriostasis rate of 74.71% (FIG. 4).

TABLE 1 counter experiment statistics

Example 2: optimization of Trichoderma atroviride T3 fermentation conditions

1. Method of

1.1 Effect of culture temperature on Trichoderma atroviride T3

50mL PDA medium was dispensed in 250mL Erlenmeyer flasks and inoculated with Trichoderma atroviride T3 spore suspension (1X 10) at 5% inoculum size ⁶ Each mL) was cultured at 22℃at 24℃at 26℃at 28℃and in a shaker at 30℃at 180r/min for 5d. Sucking 0.5mL of clear liquid, diluting to prepare spore suspension, and measuring the spore yield by using a blood cell counting plate, wherein the calculation formula is as follows: spore count/mL = average spore count per cell x 4 x 10 ⁶ X dilution.

1.2 Effect of the rotation speed on Trichoderma atroviride T3

50mL PDA medium was dispensed into 250mL Erlenmeyer flasks and inoculated with 5% of the Trichoderma T3 spore suspension (1X 10) ⁶ And (3/mL), culturing at a rotation speed of 120r/min, 140r/min, 160r/min, 180r/min, 200r/min for 5d at 28 ℃, and measuring the spore yield. .

1.3 Effect of inoculum size on Trichoderma atroviride T3

50mL PDA culture medium was dispensed into 250mL Erlenmeyer flasks and inoculated with Trichoderma atroviride T3 spore suspension (1X 10) at 5%, 10%, 15%, 20%, 25% of the inoculum size, respectively ⁶ and/mL), shaking culture was performed at 28℃and 180r/min for 5d. And measuring the spore yield. Three replicates were set for each of the above-described groups.

1.4 orthogonal experiments

L9 (3) was carried out using the culture temperature (factor A), the rotation speed (factor B) and the inoculum size (factor C) as main fermentation influencing factors and the spore production amount of Trichoderma atroviride as an index ⁴ ) Orthogonal experiments were repeated 3 times per experiment. The experimental levels of the factors are shown in Table 2. The analysis of the orthogonal experimental results adopts SPSS software.

TABLE 2 level of orthogonal test factors

2. Results

2.1 Effect of temperature on Trichoderma atroviride T3

The test results of the different culture temperatures on the spore yield of the trichoderma atroviride T3 show (figure 5), along with the increase of the temperature, the spore yield of the trichoderma atroviride T3 is increased and then reduced, and the spore yield of the trichoderma atroviride T3 reaches the highest at 28 ℃ and is 1.201 multiplied by 10 ⁷ And each mL. Therefore, the optimal spore-forming temperature of Trichoderma atroviride T3 was 28 ℃.

2.2 influence of the rotational speed on Trichoderma atroviride T3

Tests of different rotational speeds on the spore yield of Trichoderma atroviride T3 (FIG. 6) show that the spore yield is gradually increased between 120r/min and 160r/min, and the spore yield is highest at 180r/min and is 2.783 multiplied by 10 ⁷ And then declined. Because ofThe optimal spore production speed of Trichoderma atroviride T3 is 180r/min.

2.3 Effect of inoculum size on Trichoderma atroviride T3

Tests of different inoculum sizes on the spore amount of Trichoderma atroviride T3 show (FIG. 7), the spore amount of Trichoderma atroviride T3 tends to increase and decrease with increasing inoculum size, wherein the spore amount of Trichoderma atroviride T3 is highest at 10% inoculum size and is 1.977 ×10 ⁷ And each mL. Thus, the optimal sporulation inoculum size of Trichoderma atroviride T3 was 10%.

2.4 orthogonal test optimizing Trichoderma atroviride T3 liquid fermentation conditions

The extremely poor analysis result shows that the primary and secondary orders of the influence of 3 factors on the spore yield of fermentation are as follows: temperature (A)>The rotation speed (B) > inoculum size (C), wherein the fermentation temperature is 30 ℃, the rotation speed is 180r/min, and the inoculum size is 10%, when the Trichoderma atroviride T3 sporulation is highest, the optimal combination is A ₁ B ₁ C ₁ (Table 3). Analysis of variance results show that the influence of each factor on the spore yield of trichoderma atroviride T3 liquid fermentation is extremely remarkable in fermentation temperature influence (P<0.01 The effect of the rotation speed and the inoculation amount is not significant (P>0.05 (table 4).

Table 3 (L) ₉ (3) ⁴ ) Orthogonal test design and range analysis

Table 4L ₉ (3 ⁴ ) Analysis of variance of orthogonal test

Example 3: potted plant prevention effect determination of trichoderma atroviride T3 fermentation liquor on coptis root rot

1. Method of

Selecting healthy coptis plants with consistent height and growth vigorSoaking the Chinese goldthread in sodium hypochlorite solution with the mass percent concentration of 5% for 3min, washing the Chinese goldthread with sterile water for 3 times, and planting the Chinese goldthread in a sterile nutrient medium (high-temperature sterilization) of the flowerpot, wherein each pot contains 1 plant, and the total number of the Chinese goldthread is 20 pots. Trichoderma atroviride T3 was cultured under optimal fermentation conditions selected in the orthogonal experiment of example 2 to give a concentration of 1X 10 ⁶ A spore suspension of Fusarium oxysporum Coptis HL-05 (1×10) was also prepared at the same time ⁶ and/mL). Root-irrigating coptis chinensis with 5mL spore suspension of fusarium oxysporum HL-05. After 24h, the trichoderma atroviride T3 spore suspension was applied by root irrigation, 5mL each, for a total of 10 pots. As a control, 10 pots were used to which 5mL of sterile water was applied. After 20d, the morbidity, disease index and control were observed and calculated.

The incidence degree of the coptis root rot is according to a 5-level grading standard, and the incidence condition is counted.

Level 0: the plant leaves are complete and healthy, the root system is healthy, and no disease occurs.

Stage 1: plants with 1-3 leaves showed a green-loss condition, and root systems did not develop.

2 stages: more than 4 leaves of the plant turn yellow, the disease spots appear, and the root system turns black.

3 stages: most of the leaves of the plants turn yellow or wilt and fall, the root system becomes black and soft rot, and the stem base is severely soft rot.

4 stages: the plants die, the leaves dry up, and the root system decays.

The calculation method comprises the following steps:

incidence (%) = number of diseased plants/total number of plants x 100;

disease index = Σ (number of disease plants at each stage×number of disease stages)/(highest stage×total number of investigation) ×100;

relative control effect (%) = (control disease index-treatment disease index)/control disease index x 100.

2. Results

The healthy coptis plants are subjected to root irrigation treatment by fusarium oxysporum, and after 10 days, leaves begin to appear yellow brown, the incidence rate of a control group is 100% in 20 days, the effect of trichoderma atroviride T3 on preventing and treating the coptis root rot is remarkable, and the incidence rate is controlled to be 30% (shown in table 5). C in control group ₁ Plants and G ₁ The disease index of the plants is grade 4; a is that ₁ Plants and F ₁ The disease index of the plants is grade 3; b (B) ₁ Plants and D ₁ The disease index of the plants is grade 2; e (E) ₁ Plants, H ₁ Plants, I ₁ Plants and J ₁ The plant disease index was grade 1 (as shown in fig. 8). Namely, the trichoderma atroviride T3 is used for preventing and treating root rot, and the disease index is reduced from 55 to 7.5. The control efficiency is up to 86.36%.

TABLE 5 potted plant control effect of Trichoderma atroviride T3 fermentation broth on coptis root rot

Note that: CK is inoculated with fusarium oxysporum pathogen only.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

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Claims

1. The trichoderma atroviride T3 is characterized in that the preservation name of the trichoderma atroviride T3 is trichoderma atrovirideTrichoderma atroviride) The preservation address is preserved in China center for type culture Collection: the university of martial arts collection, accession number: CCTCC No. M2022330, date of preservation: 2022, 03, 28.

2. A microbial inoculum for preventing and treating root rot of coptis, which is characterized by being a spore suspension prepared by fermenting and culturing trichoderma atroviride T3 according to claim 1.

3. The microbial inoculum for preventing and treating root rot of coptis according to claim 2, wherein the pathogenic bacteria of the root rot of coptis are fusarium putrescens, fusarium oxysporum and fusarium tricornutum.

4. The method for preparing the microbial inoculum for preventing and treating root rot of coptis, which is characterized by comprising the following steps:

taking Trichoderma atroviride T3 strain, picking a small amount of mycelia under aseptic condition, inoculating into sterilized PDA culture medium, dark culturing at 30deg.C and rotation speed of 180r/min for 5-6d, sucking clear liquid, and diluting to obtain spore suspension.

5. Use of a microbial agent for preventing and treating root rot of coptis as claimed in claim 2, wherein the spore suspension is applied to root irrigation of coptis plants.

6. The use of a microbial agent for preventing and treating root rot of coptis as set forth in claim 5, wherein the concentration of the spore suspension is 0.8 to 1X 10 ⁶ And each mL.