CN113755340B - Trichoderma harzianum and application thereof - Google Patents

Abstract

The present invention provides a Trichoderma harzianum-GXMD-hs-g5, which was deposited at the Guangdong Microbial Culture Collection Center on March 4, 2021 with a preservation number of GDMCC NO: 61869. Easy to save. The invention overcomes the shortcomings of the existing technology, has reasonable design and compact structure. It is proved through experiments that Trichoderma harzianum-GXMD-hs-g5 can coexist with citrus root systems to form mycorrhizae and significantly promote the growth of citrus. Effect. The discovery of the strain has enriched my country's available microbial resources. Its growth-promoting effect is stable, efficient, and environmentally friendly, and it has good application prospects in citrus cultivation.

Description

A kind of Trichoderma harzianum and its application

Technical field

The present invention relates to the field of microbial technology, and specifically to a kind of Trichoderma harzianum and its application.

Background technique

Complex problems such as topography and topography in citrus planting areas lead to large differences in land fertility distribution and poor targeted fertilization effects, resulting in low orchard yields, reduced fruit quality and many other problems. Farmers often apply fertilizer blindly to this problem, exacerbating the problem of chemical fertilizers in orchards. The occurrence of pollution problems affects the sustainable development of the citrus industry. Citrus root hairs are few and short, and soil that is too dry or too wet will have a serious impact on citrus.

Mycorrhizal symbiosis helps to support and fix plants, supply water and oxygen, improve plant nutritional status, and can also help plants absorb mineral nutrients in the soil, improving plants' ability to survive in metal environments, saline-alkali zones, high temperature droughts, low temperature freezing, water It can improve the stress resistance of flooded environments, enhance plant disease resistance, and play an important role in resisting invasive plants and restoring ecosystems.

During the development of plant root systems, if a good mycorrhizal structure can be formed with suitable mycorrhizal fungi, yields can be increased and quality improved. Arbuscular mycorrhizas help plants resist adverse environmental stress and pests and diseases, promote healthy plant growth, and can reduce the amount of chemical fertilizers and pesticides used to reduce the amount of chemical substances that are harmful to the environment and ecology. Arbuscular mycorrhizal symbionts can accelerate root growth, improve the absorption of low-mobility inorganic ions, accelerate nutrient recycling, enhance plant tolerance to adverse stress (biotic and abiotic) factors, form a good soil structure, and improve Diversity of plant groups.

In order to increase the yield of citrus, we propose a Trichoderma harzianum and its application.

Contents of the invention

The purpose of the present invention is to solve or at least alleviate the problems existing in the prior art.

In order to achieve the above objectives, the present invention is achieved through the following technical solutions:

A kind of Trichoderma harzianum-GXMD-hs-g5, which was deposited at the Guangdong Microbial Culture Collection Center on August 12, 2021 with the deposit number GDMCC NO: 61869.

Optionally, the storage method of the Trichoderma harzianum-GXMD-hs-g5 is to store it on a slope in a test tube and store it in a refrigerator at 4°C, and the culture medium used is PDA culture medium.

Optionally, the sampling location of the citrus root system of Trichoderma harzianum-GXMD-hs-g5 is the Wogan field in Jiangxi Town, Xixiangtang District, Nanning City, Guangxi. The topsoil is removed and the roots of Wogan are collected.

Optionally, a method for isolating and purifying Trichoderma harzianum includes the following steps:

Step 1. Material preparation; sample sampling: The citrus root system was sampled at the Wogan field in Jiangxi Town, Xixiangtang District, Nanning City, Guangxi. The topsoil was removed, the root system of Wogan was collected, preserved with FAA fixative, and brought back to the laboratory for the next step. deal with;

Step 2: Prepare isolation and purification medium:

Step 3. Isolation and purification of strains

Rinse the collected tangerine root system with clean water and cut it into 2~4cm sections; first wash it with sterile water 5~6 times in a super clean bench, then disinfect it with 75% alcohol for 30 seconds, and continue to rinse it with sterile water for 5 seconds. ~6 times, then disinfect with 0.1% mercury chloride for 1~10 minutes, set several time gradients, and finally rinse with sterile water 5~6 times; cut the disinfected root system into 0.5cm sections and place them on the PDA culture medium , cultured in a 30°C incubator;

Pick the fringe hyphae of the fungus growing on the PDA medium, inoculate it into a new PDA medium for purification, and subculture until the strain is purified.

Optionally, the preparation, isolation and purification medium in step two is PDA medium and PDB medium; the formula of PDA medium includes the following parts by weight: 200.0g potato, 20.0g glucose, 20.0g agar, and 1000mL distilled water;

The formula of PDB medium includes the following weight parts: 200.0g potato, 20.0g glucose, and 1000mL distilled water.

Alternatively, a method for preparing fermentation broth of Trichoderma harzianum-GXMD-hs-g5 is to transfer the preserved strain to a PDA culture medium plate with an inoculation loop and place it in an incubator at 28~32°C Cultivation, after the strain has grown to cover half of the plate area, use an inoculation loop to transfer it to the sterilized PDB culture medium, place it in a shaking shaker at 28~32°C, 150~200r/min, and culture for 2~3 days. .

Optionally, the application of a Trichoderma harzianum fermentation broth to promote the growth of Citrus aurantium seedlings includes the following steps:

Slow down the seedlings: Transplant the experimental seedlings of Citrus aurantium into a flower pot filled with soil + substrate (1:1) to ensure that the original soil nutrients are relatively consistent;

Seedling selection: After slowing down the seedlings for a period of time, select Citrus aurantium seedlings with relatively consistent growth to carry out experiments; make an experimental group and a control group, wherein the experimental group is irrigated with the strain fermentation liquid provided by the invention, and the control group is cultured with an equal amount of PDB liquid Water the base; after marking, place them randomly;

Watering of bacteria: In the experimental group, each experimental seedling was poured with 5 ml of strain fermentation broth, and in the control group, each experimental seedling was poured with 5 ml of PDB liquid culture medium; once every two weeks;

Measurement: Use rulers, vernier calipers, etc. to measure the plant height, ground diameter and other indicators of the experimental seedlings; measure the initial data before watering the fungicides, measure the relevant indicators again two months later, and pull out the experimental seedlings to measure the root length.

The embodiment of the present invention provides Trichoderma harzianum and its application. It has the following beneficial effects: the strain is simple to screen and easy to cultivate, and the inventor has also established a corresponding culture method. Experiments have shown that Trichodermaharzianum-GXMD-hs-g5 can symbiosis with citrus roots, form mycorrhizae, and have a significant growth-promoting effect on citrus. The discovery of the strain has enriched my country's available microbial resources. Its growth-promoting effect is stable, efficient, and environmentally friendly, and it has good application prospects in citrus cultivation.

Description of drawings

Figure 1 is a diagram of the growth morphology of Trichoderma harzianum on the third day on PDA medium;

Figure 2 is a diagram of the growth morphology of Trichoderma harzianum on the PDA medium on the seventh day;

Figure 3 is a diagram of the root system of citrus roots infected by Trichoderma harzianum under a microscope;

Figure 4 is a bar chart of the plant height increment of Citrus aurantium seedlings;

Figure 5 is a bar chart of the increment of ground diameter of Citrus aurantium seedlings;

Figure 6 is a column chart of the incremental number of leaves of Citrus aurantium seedlings;

Figure 7 is a bar chart of root length of Citrus aurantium seedlings.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Example 1

The present invention discloses a Trichoderma harzianum-GXMD-hs-g5, which was deposited at the Guangdong Microbial Culture Collection Center on August 12, 2021 with the deposit number GDMCC NO: 61869.

The preservation method of Trichoderma harzianum-GXMD-hs-g5 is to store it on the slope of the test tube and store it in the refrigerator at 4°C. The culture medium used was PDA culture medium.

The sampled citrus root system of Trichoderma harzianum was taken from the Wogan field in Jiangxi Town, Xixiangtang District, Nanning City, Guangxi. The topsoil was removed and the roots of Wogan were collected.

Example 2

The isolation and purification method of Trichoderma harzianum-GXMD-hs-g5 includes the following steps

Step 1. Material preparation

Sample sampling: The citrus root system was sampled at the Wogan field in Jiangxi Town, Xixiangtang District, Nanning City, Guangxi. The topsoil was removed, and the root system of Wogan was collected. FAA fixative (38% formaldehyde 5ml, glacial acetic acid 5ml, 70% alcohol 90ml) was used. Save and bring back to the laboratory for further processing.

Step 2: Prepare isolation and purification medium:

PDA medium: 200.0g potato, 20.0g glucose, 20.0g agar, 1000mL distilled water.

PDB medium: 200.0g potato, 20.0g glucose, 1000mL distilled water.

Step 3. Isolation and purification of strains

Rinse the collected tangerine root system with clean water and cut it into 2~4cm sections. First wash with sterile water for 5 to 6 times in a ultra-clean station, then disinfect with 75% alcohol for 30 seconds, continue to rinse with sterile water for 5 to 6 times, and then disinfect with 0.1% mercury chloride for 1 to 10 minutes. Set several times. Gradient, and finally rinse with sterile water 5 to 6 times. Cut the sterilized root system into 0.5cm sections and place them on PDA culture medium, and culture them in a 30°C incubator.

Pick the fringe hyphae of the fungus growing on the PDA medium, inoculate it into a new PDA medium for purification, and subculture until the strain is purified.

Example 3

Preparation of the Trichoderma harzianum-GXMD-hs-g5 fermentation broth

Use an inoculation loop to transfer the preserved strain to the PDA culture medium plate, and place it in an incubator at 28~32°C for cultivation. After the strain has grown to cover half of the plate area, use an inoculation loop to transfer it to the sterilized PDB culture medium. , place it in a shaker at 28~32℃, 150~200r/min and shake for 2~3 days.

Experimental example 1

Determination of Trichoderma harzianum-GXMD-hs-g5 infection of citrus root systems

Experiments were carried out using Citrus aurantium seedlings to determine the root infection rate of the strains. Plant the Citrus aurantium seedlings in flower pots containing high-temperature sterilized substrate, and place them in a culture room to slow down the seedlings. The Trichoderma harzianum provided by the present invention is cultured in a PDB medium. After forming a bacterial ball, the experimental seedlings are subjected to root irrigation treatment, and each experimental seedling is irrigated with 5 ml of the above bacterial agent. After one month, the experimental seedlings were pulled out, and their roots were used to measure the infection rate of the strains on the roots of the Citrus aurantium seedlings.

The acetic acid ink staining method was used to determine the infection rate of the roots of Citrus aurantium seedlings by the strains. Rinse the root system with sterile water, cut it into 0.5cm sections, place it in a 5% acetic acid ink solution containing pure white vinegar in a 100°C water bath for 3 minutes, clean and decolorize with water or white vinegar, and use it for microscopic examination. Select 100 sections of dyed roots and observe under a microscope whether the roots are infected by fungi and form mycorrhizae. The formula for calculating the infestation rate is as follows:

Experimental results show that the infection rate of the Trichoderma harzianum provided by the present invention on the root system of Citrus aurantium seedlings is as high as 47%. The mycorrhizal diagram under the microscope is shown in Figures 1-2. Figure 1 shows the bacterial strain cultured on PDA at 3 days. Regarding the morphology on the base, Figure 2 shows the morphology of the strain on PDA medium at 7 days, and Figure 3 shows the infected root system under a microscope.

Experimental example 2

The application of Trichoderma harzianum-GXMD-hs-g5 to Citrus aurantium seedlings

Slow down the seedlings: Transplant the experimental seedlings of Citrus aurantium into flower pots filled with soil + substrate (1:1) to ensure that the original soil nutrients are relatively consistent.

Seedling selection: After slowing down the seedlings for a period of time, select Citrus aurantium seedlings with relatively consistent growth patterns to conduct experiments. An experimental group and a control group were formed. The experimental group was irrigated with the strain fermentation liquid provided by the invention, and the control group was irrigated with an equal amount of PDB liquid culture medium. After marking them, place them randomly.

Pouring bacteria: Each experimental seedling in the experimental group was poured with 5 ml of strain fermentation broth, and each experimental seedling in the control group was poured with 5 ml of PDB liquid culture medium. Water every two weeks.

Measurement: Use a ruler, vernier caliper, etc. to measure the plant height, ground diameter and other indicators of the experimental seedlings. Before watering the inoculants, the initial data were measured. Two months later, the relevant indicators were measured again, and the experimental seedlings were pulled out to measure the root length.

Experimental results show that the strain fermentation liquid provided by the invention has obvious growth-promoting effect on Citrus aurantium seedlings. At the same time, Citrus aurantium seedlings are commonly used rootstocks for citrus. Citrus aurantium is also a variety of citrus, so it will also increase the promotion effect on citrus. In two months, the Citrus aurantium seedlings irrigated with the fermentation liquid of the above strains were compared with the Citrus aurantium seedlings in the control group. The plant height of the seedlings increased by 47.29%, the ground diameter increased by 19.82%, and the number of leaves increased by 40.76%. In addition, the fermentation broth of the above strains also increased the root length of Citrus aurantium seedlings by 3.81% compared with the control group. The experimental results are shown in Figure 4-7.

Experimental example 3

Strain genetic identification

To sequence the strain, the specific steps are as follows: Use CTAB method to extract strain DNA, pick out a few mycelium, grind it with liquid nitrogen, soak the ground mycelium in 0.7ml DNA extraction solution, add 0.7ml chloroform-isoamyl after 30 minutes in water bath Alcohol mixture (24/1), shake to make it turbid, centrifuge at 12000/min for 15 minutes to get the supernatant, add 1ml of DNA precipitation buffer to the supernatant, precipitate at room temperature for 30 minutes, centrifuge at 12000/min for 15 minutes to get the precipitate, add Place 0.5 ml NaCl solution and 2 times the volume of absolute ethanol in the refrigerator for 30 min, centrifuge at 12,000/min for 15 min to remove the precipitate, wash the precipitate with 1 ml of 70% alcohol and centrifuge at 12,000/min for 15 min. Repeat three times to obtain DNA precipitate. After using PCR technology to amplify the target fragment for sequencing, the extracted strain DNA was sequenced by Wuhan Aoke Dingsheng Biotechnology Co., Ltd. and the resulting sequence was matched on NCBI (the official website of the National Center for Biotechnology Information).

Trichoderma harzianum-GXMD-hs-g5 18S rRNA gene sequence: Sequence Listing

<110> Guangxi Lvyounong Biotechnology Co., Ltd.

Guangxi University for Nationalities

<120> A kind of Trichoderma harzianum and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 418

<212> RNA

<213> Trichoderma harzianum-GXMD-hs-g5

<400> 2

aggaacgacc aaacggcccg gcgggaccgc cccggggcgc gcagccccgg accaaggcgc 60

ccgccggagg accaaccaaa accgaacccc ccgcgggaaa cgagccccgg cgccccgagg 120

cgcgaaaaga acaaaaccaa caacggaccg gcggcacgag aagaacgcag cgaaagcgaa 180

agaaggaagc agaacaggaa cacgaacgaa cgcacagcgc ccgccagacg gcgggcagcc 240

gccgagcgca caaccccgaa ccccccgggg ggcggcgggg gacggcccgc ccggcggggc 300

cgcccgaaaa cagggcggcc gccgcagccc ccgcgcagag gcacaccgca cgggagcgcg 360

gcgcgccaca gccgaaacac ccaaccgaaa ggacccggac aggaggaaac ccgcgaac 418

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that these entities or operations are mutually exclusive. any such actual relationship or sequence exists between them. Furthermore, the terms "comprises," "comprises," or any other variations thereof are intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also those not expressly listed other elements, or elements inherent to the process, method, article or equipment. Without further limitation, an element defined by the statement "comprises a..." does not exclude the presence of additional identical elements in a process, method, article, or apparatus that includes the stated element.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the foregoing embodiments. The recorded technical solutions may be modified, or some of the technical features thereof may be equivalently replaced; however, these modifications or substitutions shall not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims (2)

1. Trichoderma harzianumTrichoderma harzianum) It was deposited on month 8 and 12 of 2021 under accession number GDMCC NO:61869, deposited in the Cantonese province microorganism strain collection.

2. The use of trichoderma harzianum fermentation broth according to claim 1 for promoting the growth of seedlings of bitter orange, comprising the steps of:

and (3) seedling reviving: transplanting the experimental seedlings of the fructus aurantii into a flowerpot filled with soil and matrix, so as to ensure that the original soil has relatively consistent nutrition;

selecting seedlings: after the seedlings are slowly grown for a period of time, selecting the bitter orange seedlings with relatively consistent growth vigor to carry out experiments; the method comprises the steps of (1) preparing an experimental group and a control group, wherein the experimental group is irrigated by strain fermentation broth, and the control group is irrigated by PDB liquid culture medium with equal quantity; after marking, randomly placing;

and (3) fungus watering: 5ml of strain fermentation liquor is poured into each experimental seedling of the experimental group, and 5ml of PDB liquid culture medium is poured into each experimental seedling of the control group; once every two weeks;

measurement: measuring plant height and ground diameter indexes of experimental seedlings by using a ruler and a vernier caliper; before the strain fermentation broth is irrigated, initial data are measured, relevant indexes are measured again after two months, and experimental seedlings are pulled out to measure the root length.