阅读说明：本技术 一种用于防治压砂西瓜枯萎病的木霉复合生物菌剂及其应用 (Trichoderma composite biological agent for preventing and treating blight of casserole watermelon and application thereof ) 是由 康萍芝 白小军 杜玉宁 王喜刚 张治科 张华普 马建华 邢敏 李锋 于 2021-06-25 设计创作，主要内容包括：本发明公开了一种用于防治压砂西瓜枯萎病的木霉复合生物菌剂及其应用,属于生物防治领域。其中,所述木霉生物菌剂包括Trichoderma afroharzianum CGMCC No.22433、T.atroviride CGMCC No.22432和T.longibrachiatum CGMCC No.22434中的至少一种。本发明的木霉菌菌株产孢量大,对土传病害枯萎病的病原菌抑制作用明显,并且性状稳定,无污染,无公害,使用方法简单,价格低廉,在植株体内与寄主可形成共生关系,稳定性强,能改善土壤微生态环境,对植株促生长作用明显。施用后可降低化学农药的使用量和抗药性,明显减少死苗发生,可有效缓解压砂瓜连作障碍,达到提质增效的目的,促进压砂瓜产业的绿色发展,适合推广应用。(The invention discloses a trichoderma composite biological agent for preventing and treating fusarium wilt of casserole watermelon and application thereof, belonging to the field of biological prevention and treatment. Wherein the trichoderma biological agent comprises Trichoderma afroharzianum CGMCC No.22433、 T.atroviride CGMCC No.22432 and T.longibrachiatum at least one of CGMCC No. 22434. The trichoderma strain has large spore yield, obvious inhibition effect on pathogenic bacteria of soil-borne diseases blight, stable character, no pollution, no public nuisance, simple use method, low price, strong stability, capability of forming symbiotic relationship with hosts in plants, capability of improving soil micro-ecological environment and obvious growth promotion effect on the plants. After the fertilizer is applied, the use amount and the drug resistance of chemical pesticides can be reduced, the occurrence of dead seedlings is obviously reduced, the continuous cropping obstacle of the pressed sand melons can be effectively relieved, the purposes of quality improvement and efficiency improvement are achieved, the green development of the pressed sand melon industry is promoted, and the fertilizer is suitable for popularization and application.)

1. A Trichoderma complex inoculant for controlling fusarium wilt of casserole, said Trichoderma inoculant comprising at least one of Trichoderma africanum (Trichoderma africanum), Trichoderma atroviride (Trichoderma atroviride) and Trichoderma longibrachiatum (Trichoderma longibrachiatum), wherein said at least one of: trichoderma africanum (Trichoderma africanum) was deposited at 26/5/2021 in the China general microbiological culture Collection center (CGMCC) with the following deposit numbers: CGMCC No. 22433; the Trichoderma atroviride (Trichoderma atroviride) is preserved in China general microbiological culture collection center (CGMCC) at 26.5.2021, with the preservation number as follows: CGMCC No. 22432; the Trichoderma longibrachiatum (Trichoderma longibrachiatum) is preserved in China general microbiological culture Collection center (CGMCC) at 26.5.2021, and the preservation number is as follows: CGMCC No. 22434.

2. The trichoderma complex inoculant according to claim 1, wherein said trichoderma complex inoculant comprises at least two of said three trichoderma species.

3. The trichoderma composite microbial inoculant of claim 2, wherein said trichoderma composite microbial inoculant comprises said three trichoderma fungi.

4. The complex trichoderma microbial inoculant according to any one of claims 1 to 3, further comprising an auxiliary agent.

5. The complex trichoderma inoculant according to claim 4, wherein the auxiliary agents comprise at least one of diatomaceous earth and wheat bran.

6. The complex trichoderma inoculant according to any one of claims 1 to 3, further comprising a synergist.

7. The complex trichoderma inoculant of claim 6, wherein the potentiator comprises at least one of chitosan and fulvic acid.

8. The trichoderma composite biological agent according to claim 1, which comprises t.afroharibanum CGMCC No.22433, t.atroviride CGMCC no 22432 and t.longibracaattatum CGMCC no 22434, and further comprises diatomaceous earth, wheat bran, chitosan and fulvic acid.

9. The Trichoderma complex microbial agent of claim 8, wherein the weight ratio of T.afrorazaanum CGMCC No.22433, T.atroviride CGMCC No.22432 and T.longorachiatum CGMCC No.22434 is 3:2:1, the ratio of Trichoderma is 75%, and the effective spore number is not less than 10¹⁰CFU/g, 3-10% of diatomite, 10-20% of wheat bran, 1-3% of chitosan and 1-5% of biochemical fulvic acid.

10. The trichoderma complex inoculant according to claim 9, wherein the t.afroharibanum CGMCC No.22433, t.atroviride CGMCC no 22432 and t.longbrachThe weight ratio of iatum CGMCC No.22434 is 3:2:1, the ratio of trichoderma is 75%, and the effective spore number is not less than 10¹⁰CFU/g, 6% of diatomite, 12% of wheat bran, 3% of chitosan and 4% of biochemical fulvic acid.

Technical Field

The invention belongs to the field of biological control, and particularly relates to a trichoderma composite biological agent for controlling fusarium wilt of watermelon sand pressing and application thereof.

Background

The sand-pressing melon industry is a new pillar industry for farmers to defy poverty and become rich, increase income and reduce disasters in arid areas in the middle of Ningxia, and the existing sand-pressing land is 90 ten thousand mu. Due to the special geographical environment conditions and planting modes of the sand compaction land, the main planting mode is continuous cropping watermelon. With the increase of the planting age of the sand-pressing land and the continuous cropping planting, ecological suitability and pathogen accumulation of the sand-pressing melons, favorable conditions are created for the outbreak and the prevalence of diseases, and the monitoring and control technology is lagged behind, so that the sand-pressing land is degraded year by year, and especially the soil-borne disease sand-pressing melon fusarium wilt or continuous cropping obstacle becomes the most prominent and troublesome problem to control. Through investigation, the soil-borne disease of the sand pressing melon is serious in the disease of a land where the sand pressing is carried out for more than 7 years since 2010, the plant death rate reaches 10% -40%, and the serious plant destruction becomes a main bottleneck for restricting the continuous development of the sand pressing land, so that the brand quality and the sustainable and healthy development of the industry of the Ningxia sand pressing melon are seriously influenced. In addition, the unreasonable use of chemical fertilizers and pesticides also causes a plurality of problems of soil recession of the sand compaction land, serious environmental non-point source pollution and the like, so that the yield of sand compaction melons is reduced, the quality is reduced, and the benefit is lowered.

Trichoderma sp fungi are widely distributed worldwide, are one of important communities of soil microorganisms, are plant endophytic fungi, and can be separated from soil, plant rhizosphere, stem, leaf, seed and corm surfaces, plant residues such as dry branches and fallen leaves, and fruiting bodies of other fungi. The existing research data show that trichoderma has wide application value in the aspects of agriculture, industry, environmental protection and the like, the application performance of the trichoderma in the agricultural field is particularly outstanding, and various products such as trichoderma biocontrol inoculants, biological bacterial fertilizers, resistance inducers, seed coating agents and the like are registered and commercially produced and play an important role in agricultural production. Among them, Trichoderma harzianum (Trichoderma harzianum) is mainly abundant, and Trichoderma viride (Trichoderma viride) is secondly abundant. The following patents (applications) related to trichoderma in recent years mainly include: CN101928673A discloses a biocontrol strain trichoderma harzianum, and the bacteriostatic action on several pathogenic bacteria is measured indoors; CN101693879A discloses a Trichoderma longibrachiatum strain, wherein wheat bran is used as a solid fermentation culture medium for preparation of a microbial inoculum, and diseases and the like of bark of trees are subjected to prevention and effect measurement; CN101574091A discloses a wettable powder of high spore powder of trichoderma and a preparation method thereof, and provides a preparation method of wettable powder of trichoderma viride; and so on. Compared with trichoderma viride, the research of the endophytic trichoderma viride starts late, related reports are few, but the endophytic trichoderma viride can be symbiotic with host plants, has obvious advantages in disease resistance, stress resistance, stability and the like, has a plurality of advantages of strong disease resistance and stress resistance and the like, is more and more valued by researchers at present, and has huge development and application potentials. CN106520570A discloses an endophytic hook trichoderma strain CCTCC NO: m2016366, the strain has broad antibacterial spectrum, shows antibacterial activity against 5 plant pathogenic bacteria, has strongest inhibition effect against Magnaporthe grisea, and can be used for preventing and treating plant diseases. However, no application of endophytic trichoderma in preventing and treating blight of watermelon with sand pressing exists at present.

Disclosure of Invention

The invention aims to overcome the current situations of low control effect, poor stability and the like of a single trichoderma composite biological agent on the blast disease of the sand-pressed watermelon in the prior art, can more effectively solve the problems of the blast disease and continuous cropping obstacles in the prior art, and has more obvious growth promotion effect on the sand-pressed watermelon.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a Trichoderma complex inoculant for controlling fusarium wilt of casserole, comprising at least one of Trichoderma africanum (Trichoderma africanum), Trichoderma atroviride (Trichoderma atroviride) and Trichoderma longibrachiatum (Trichoderma longibrachiatum), wherein said Trichoderma viride comprises: trichoderma africanum (Trichoderma africanum) was deposited at 26/5/2021 in the China general microbiological culture Collection center (CGMCC) with the following deposit numbers: CGMCC No.22433, the preservation address is: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the Trichoderma atroviride (Trichoderma atroviride) is preserved in China general microbiological culture collection center (CGMCC) at 26.5.2021, with the preservation number as follows: CGMCC No.22432, the preservation address is: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North; the Trichoderma longibrachiatum (Trichoderma longibrachiatum) is preserved in China general microbiological culture Collection center (CGMCC) at 26.5.2021, and the preservation number is as follows: CGMCC No.22434, the preservation address is: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

In some embodiments of the invention, the trichoderma complex inoculant comprises at least two of the three trichoderma species. In some preferred embodiments of the present invention, the trichoderma complex inoculant comprises the three trichoderma species.

In some embodiments of the invention, the trichoderma complex inoculant further comprises an adjuvant. In some preferred embodiments of the present invention, the adjuvant comprises at least one of diatomaceous earth and wheat bran. In some embodiments of the invention, the adjuvant consists of diatomaceous earth and wheat bran.

In some embodiments of the invention, the trichoderma complex inoculant further comprises a synergist. In some preferred embodiments of the present invention, the synergist comprises at least one of chitosan and fulvic acid. In some embodiments of the invention, the potentiator consists of chitosan and fulvic acid.

In some embodiments of the invention, the trichoderma complex microbial inoculant comprises t.afroharizanum CGMCC No.22433, t.atroviride CGMCC no 22432 and t.longibrachitum CGMCC no 22434 or a fermentation thereof, further comprising diatomaceous earth, wheat bran, chitosan and fulvic acid.

In some preferred embodiments of the invention, the weight ratio of the T.afroharizanum CGMCC No.22433, the T.atroviride CGMCC No.22432 and the T.longibracanthus CGMCC No.22434 is 3:2:1, the Trichoderma is 75%, and the effective spore number is not less than 10¹⁰CFU/g, 3-10% of diatomite, 10-20% of wheat bran, 1-3% of chitosan and 1-5% of biochemical fulvic acid.

In some embodiments of the invention, the weight ratio of the T.afroharizanum CGMCC No.22433, the T.atroviride CGMCC No.22432 and the T.longibracanthus CGMCC No.22434 is 3:2:1, the trichoderma accounts for 75%, and the effective spore number is not less than 10¹⁰CFU/g, 6% of diatomite, 12% of wheat bran, 3% of chitosan and 4% of biochemical fulvic acid.

The invention has the advantages of

Compared with the prior art, the invention has the following beneficial effects:

the trichoderma strain has large spore yield, strong colonization in roots and plants of crops, obvious inhibition effect on pathogenic bacteria of soil-borne diseases and blight through various action mechanisms such as competition, heavy parasitism and the like, strong stability, capability of forming symbiotic relation with hosts in plants, capability of improving soil micro-ecological environment and obvious growth promotion effect on the plants.

The assistant of the trichoderma composite biological agent is diatomite and wheat bran, and the synergist is chitosan and biochemical fulvic acid, wherein the synergist can induce crops to improve disease resistance and improve soil.

The trichoderma composite biological agent has stable properties, no pollution, no public nuisance, simple use method, low price, obvious effects of preventing diseases, promoting growth and increasing yield, has the effect of promoting growth of the pressed watermelon plants, can reduce the use amount and the drug resistance of chemical pesticides after application, obviously reduces dead seedlings, can effectively relieve the continuous cropping obstacle of the pressed watermelon, achieves the purposes of improving quality and efficiency, promotes the green development of the pressed watermelon industry, and is suitable for popularization and application.

Drawings

FIG. 1 shows molecular species identification of the T2 strain as a clade.

FIG. 2 shows molecular species identification of the T15 strain as a clade.

FIG. 3 shows molecular species identification of the T6 strain as a clade.

FIG. 4 shows the disease prevention and growth promotion effects of Trichoderma complex microbial agents in a large-area test.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 isolation and characterization of endophytic Trichoderma

The inventor utilizes a conventional tissue isolation method to isolate three endophytic trichoderma fungi from melon and vegetable plants: t2, T15 and T6. Identified as Trichoderma africanum (Trichoderma africanum), Trichoderma atroviride (Trichoderma atroviride), Trichoderma longibrachiatum (Trichoderma longibrachiatum), respectively, and the molecular identification of the evolutionary tree analysis is shown in FIGS. 1, 2 and 3, respectively.

Three strains of bacteria are preserved in China general microbiological culture Collection center (CGMCC) at 26 months and 5 months in 2021, and the obtained preservation numbers are divided into: CGMCC No.22433, CGMCC No.22432 and CGMCC No. 22434. i.e.T.afroharizia africana Trichoderma harzianum, T.atroviride Trichoderma atroviride and T.longibrachiatum Trichoderma longibrachiatum.

Example 2 field test Effect of different endophytic Trichoderma

1. Determination of colonization Capacity

A pot experiment was used. Taking the pressed sand field and continuously planting soil, putting the soil into flowerpots, setting 6 treatments in total, repeating the treatment for 3 times, transplanting 1 watermelon seedling in each pot, and setting no pesticide (clear water) as a control. The pesticide application method is a hole application method, wherein two doses of trichoderma reesei microbial inoculum are respectively set to be 3 g/strain and 5 g/strain, and watermelon seedlings are uniformly mixed with soil before being transplanted. Normally managing the transplanted watermelon seedlings, digging out the plants at 20d, respectively taking the rhizosphere soil of the watermelon seedlings and different organs or tissues of the plants, and measuring the quantity of the soil and the endophytic trichoderma by using a selective culture medium and adopting a dilution flat plate method and a flat plate smearing method.

The results are shown in Table 1.

Table 1: determination of the colonization Capacity of different endophytic Trichoderma in watermelon plants and in soil (10)⁴CFU/g)

Table 1 results of the colonization ability assay show: the trichoderma agents T15, T2 and T6 have strong colonization ability in watermelon plants and soil, the R/S ratio is 1.62-5.71, the number of trichoderma on roots is more than that of stem bases and leaves, the T2 effect is the best, T15 and T6 have a growth promotion effect on the watermelon plants, and the appropriate dosage is 5 g/watermelon plant.

2. Determination of field control effect

The field plot experiment is provided with 3 treatments in total, the application dose is 5 g/plant, each treatment is repeated for 3 times, the treatments are arranged in groups randomly, and the conventional medicament 99% hymexazol WP and the non-application medicament (clear water) are used as controls; the application method is combining hole application and root irrigation, wherein the hole application is that the watermelon seedlings are uniformly mixed with soil before being transplanted, the dilution multiple of the irrigation roots is 200-300 times of liquid, and the liquid medicine amount of each plant is 250 ml; and (3) normally managing the transplanted watermelon seedlings, irrigating roots after the plants grow for 20 days, irrigating the roots for 1-3 times according to the severity of the disease condition in the growth period, and keeping the dosing interval for 15-20 days.

Table 2: influence of different trichoderma composite biological agents on generation and yield of watermelon blight of sand pressing

Table 2 the results of the field control effect measurements show that: the method for preventing and treating the blight of the sand melon by utilizing the combination of hole application and root irrigation of the self-developed trichoderma composite biological agent shows a certain prevention effect, the average field prevention effect reaches 71.14-76.7%, and the average yield is increased by 15.43-24.36%.

3. Demonstration effect verification of large-area test of trichoderma composite biological agent

In the field large-area experiment demonstration, a trichoderma composite biological agent is used for treatment, the application dosage is 5 g/plant, and the large-area is 667m²Treating with clear water as a control; the application method is point applicationCombining root irrigation, wherein hole application is to uniformly mix watermelon seedlings with soil before transplanting, the dilution multiple of the irrigation roots is 200-300 times of liquid, and the liquid medicine amount of each plant is 250 ml; and (3) normally managing the transplanted watermelon seedlings, irrigating roots after the plants grow for 20 days, irrigating the roots for 2 times in the growth period, and keeping the dosing interval period between 15 and 20 days.

Table 3: influence of trichoderma composite biological agent on generation and yield of watermelon blight of sand pressing

Table 4: influence of Trichoderma composite biological agent on soil microflora and fusarium oxysporum

After three trichoderma biological agents are compounded, a large-area control test is carried out on the press sand melon, and the measurement result shows that: after the compound microbial inoculum is applied by combining hole application and root irrigation (for 2 times), the compound microbial inoculum can obviously reduce the occurrence of the blight of the watermelon pressed sand, the field control effect reaches 79.31 percent, the yield is increased by 26.93 percent, and the quantity of pathogenic bacteria (fusarium oxysporum) in soil is reduced by 5.27 multiplied by 10²The number of CFU/g, soil bacteria and actinomycetes is respectively increased by 23.78 multiplied by 10⁵CFU/g、1.4×10⁴CFU/g, the number of trichoderma obviously increased by 16.86 multiplied by 10³CFU/g shows that the quantity of beneficial bacteria and biocontrol bacteria in the soil is obviously increased, the disease resistance of the watermelon plants pressed sand to blight is enhanced, and the soil microorganism flora is favorably regulated. Overall, the soil micro-ecological environment develops towards a favorable direction.

The biological agent can be rapidly propagated in watermelon plants and soil after being applied, effectively inhibits the quantity of pathogenic bacteria (fusarium oxysporum watermelon specialization type) through various action mechanisms such as competition, parasitism and antibiotics, forms symbiotic relation with hosts in the watermelon plants, has strong stability, and has stronger biocontrol effect by three kinds of endophytic trichoderma, so that the biological agent can more effectively prevent and control the blight of the fusarium oxysporum and regulate soil microflora compared with the single soil trichoderma in the prior art, and the soil micro-ecological environment can be developed towards a favorable direction; 2 kinds of added synergists can induce crops to improve disease resistance, improve soil and promote the growth and development of the crops; the product has no pollution, no public nuisance, simple use method, low price and obvious effects of preventing diseases, promoting growth and increasing yield, can achieve the purposes of improving quality and increasing efficiency, promotes the green development of the industry of the press sand melon, and is suitable for popularization and application.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.