CN113416651B - Viola variant of kiwi fruit endophytic antagonistic fungus fusarium tuberosum and application thereof - Google Patents

Abstract

The present invention provides a kiwifruit endogenous antagonistic fungus Fusarium nodosa varietal and its application. The strain J-1 is Fusarium nodosa varietal. The strain has been preserved in China Typical Culture Collection Center, the deposit number is CCTCC NO: M2021510. The present invention has the following advantages: an endophytic fungus having a strong antagonistic effect on the pathogenic bacteria Alternaria kiwifruit postharvest rot is isolated from kiwifruit fruit, and the bacterial strain is identified by morphological identification and molecular biology as Fusarium nodosa varietal Violet, this strain can grow at 20-30°C, and the optimum growth temperature is 28°C. The antagonistic bacteria J‑1 has a broad-spectrum antagonistic effect, and has good antagonistic effects on various pathogenic bacteria such as Phomopsis, Botrytis, Trichochromospora cocoa, etc., and its sterile fermented liquid has a strong thermal Stability and acid-base stability, and the sterile fermentation filtrate still has biological activity after being placed at room temperature for 45 days, and the persistence is good. Therefore, the strain has broad application prospects in the prevention and treatment of kiwi fruit postharvest rot.

Description

An endophytic antagonistic fungus Fusarium nodosa varietal in kiwifruit and its application

technical field

The invention belongs to the technical field of biological control of plant diseases, and more specifically relates to a kiwi fruit endophytic antagonistic fungus Fusarium meridmoides var. violaceum and its application.

Background technique

Kiwifruit soft rot, also known as ripe rot, is a disease of fruit caused by fungi, which is seriously harmful during postharvest storage of kiwifruit. The center of the diseased part of the fruit is depressed, and the diseased spots are mostly round or oval. The center of the diseased disease is milky white, and the surrounding area is yellow-green. Edible value. At present, kiwifruit soft rot is a serious disease phenomenon in Shaanxi, Guizhou, Sichuan, Hunan, Jiangxi and other places in my country, and the incidence rate is as high as 35%, which seriously restricts the development of kiwifruit industry in my country. The disease is caused by various pathogens such as Botryosphaeria sp., Phomopsis sp. and Alternaria alternata.

In recent years, my country's kiwifruit industry has developed rapidly, and the kiwifruit output ranks first in the world. However, the incidence of kiwifruit soft rot is increasing year by year, seriously affecting the development of kiwifruit industry in my country. At present, physical control and chemical control methods are mainly used for kiwifruit soft rot. Physical control requires a lot of manpower and material resources, and the cost is high, and the control effect on the disease is limited. Chemical control is prone to problems such as drug resistance, pesticide residues, environmental pollution and food safety. Therefore, it is urgent to find a safe and feasible control strategy to ensure the healthy and sustainable development of my country's kiwifruit industry.

Plant endophytes, also known as biocontrol bacteria, are a type of microorganisms that use plants as hosts to survive and reproduce, and establish a harmonious symbiotic relationship with host plants. According to their different types, they can be divided into three categories: endophytic fungi, endophytic bacteria and endophytic actinomycetes. Plant endophytes will form a variety of secondary biologically active substances in the process of biological metabolism. These substances have the effect similar to pesticides, and can play a role in sterilization, insecticide, disease prevention, nitrogen fixation and growth promotion. The beneficial biological effect of growth has been widely used in the biological control of plant diseases.

At present, there are still deficiencies in the control of kiwifruit soft rot, so it is urgent to develop a safe, efficient, economically feasible and sustainable biological control technology.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a kiwifruit endogenous antagonistic fungus Fusarium nodosa Viola varietal and its application, which has a strong antagonistic effect on various pathogenic bacteria such as kiwifruit fruit rot pathogenic bacteria Alternaria According to the morphological characteristics and molecular biological methods, it was identified as Fusarium meridmoides var. violaceum, and the stability of its sterile fermentation broth was determined through experiments, which can be used for the control of soft rot disease of kiwifruit.

The purpose and efficacy of a kind of endophytic fungus of Fusarium nodosa var. viola colori and its application of the present invention are achieved by the following specific technical means:

A kiwifruit endophytic antagonistic fungus Fusarium nodosa var. Viola varietal, the name of the strain is J-1, its preservation number is: CCTCC NO: M2021510, and the preservation date is: May 11, 2021.

The strain is isolated from kiwi fruit.

The application of a kiwifruit endophytic antagonistic fungus Fusarium nodosa Viola varietal, the strain has antagonistic effect on the pathogenic bacteria of kiwifruit fruit rot.

The application method of a kiwi fruit endophytic antagonistic fungus Fusarium nodosa Viola varietal, the method is as follows: strain J-1 is cultured on PDA medium and then inoculated on kiwi fruit fruit.

The application method of a kiwi fruit endophytic antagonistic fungus Fusarium nodosa var. viola. The method is as follows: preparing the aseptic fermentation filtrate of the bacterial strain J-1, and inoculating the filtrate on the kiwi fruit.

The content of the filtrate is 15-25%.

The present invention at least includes the following beneficial effects:

An endophytic fungus isolated from kiwifruit that has a strong antagonistic effect on various pathogenic bacteria such as Alternaria sp. Variant (Fusarium meridmoides var. violaceum), the aseptic fermentation filtrate of this strain has strong temperature adaptability and acid-base adaptability, and its antibacterial active substances have a long lasting effect. After the kiwi fruit is treated with the antagonistic bacteria fermentation liquid, it can effectively inhibit the infection of Alternaria spp., and the fermentation liquid has no pathogenicity to kiwi fruit, so it has potential commercial development and application value, and has a wide range of applications in the biological control of kiwi fruit rot disease. application prospects.

Description of drawings:

Fig. 1 is the morphological feature figure of antagonistic bacteria of the present invention: the yeast-like slimy layer formed in the early stage of growth on the PDA medium;

Fig. 2 is the morphological feature figure of antagonistic bacteria of the present invention: antagonistic fungus purple-red colony and crown hyphae one;

Fig. 3 is the morphological feature figure of antagonistic bacteria of the present invention: antagonistic fungus purple-red colony and crown hyphae II;

Fig. 4 is the morphological characteristic figure of antagonistic bacteria of the present invention: myxospore group;

Fig. 5 is the morphological feature figure of antagonistic bacteria of the present invention: single bottle stem sporulation structure;

Figure 6 is a morphological characteristic figure of the antagonistic bacteria of the present invention: spore micromorphological figure 1;

Fig. 7 is the morphological characteristic figure of antagonistic bacteria of the present invention: spore micromorphological figure 2;

Figure 8 is a morphological characteristic diagram of the antagonistic bacteria of the present invention: Mycelia micromorphological diagram 1;

Fig. 9 is a morphological characteristic diagram of the antagonistic bacteria of the present invention: Mycelia microscopic morphology Fig. 2;

Fig. 10 is the multigene phylogenetic tree of the present invention;

Fig. 11 is the morphological feature figure of the present invention on the PDA medium;

Fig. 12 is the morphological feature figure of the present invention on the PSA medium;

Fig. 13 is the morphological feature figure of the present invention on OA medium;

Fig. 14 is the morphological feature figure of the present invention on the CA medium;

Fig. 15 is the morphological feature figure of the present invention on the Czapek medium;

Fig. 16 is the morphological feature figure of the present invention on Bilais medium;

Fig. 17 is the plate antagonism effect diagram of the present invention to Alternaria;

Fig. 18 is the plate antagonism effect figure of the present invention to Phomopsis;

Fig. 19 is a plate antagonism effect diagram of the present invention to Botrytis spp.;

Fig. 20 is a plate antagonism effect diagram of the present invention to Cocoa Trichoderma;

Fig. 21 is the comparative figure of the inhibitory effect of fermented liquid of the present invention to Alternaria;

Figure 22 is the influence of temperature on the antibacterial activity of J-1 bacterial strain fermented liquid;

Figure 23 is the effect of pH on the antibacterial activity of J-1 strain fermented liquid;

Figure 24 is the impact of storage time on the antibacterial activity of J-1 strain fermented liquid;

Figure 25 is a comparison chart of the indoor control effect of J-1 filtrate on Alternaria.

Detailed ways

Embodiments of the present invention will be described in further detail below through examples. The following examples are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

In the description of the present invention, unless otherwise stated, "plurality" means two or more; the terms "coaxial", "bottom", "one end", "top", "middle", "other The orientation or positional relationship indicated by "one end", "upper", "one side", "top", "inner", "front", "central", "both ends" etc. is based on the orientation or position shown in the drawings The relationship is only for the convenience of describing the present invention and simplifying the description, but does not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation of the present invention. In addition, the terms "first", "second", "third", etc. are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

In the description of the present invention, it should be noted that, unless otherwise clearly stipulated and limited, terms such as "installation", "setting", "connection", "fixing", and "screwing" should be understood in a broad sense, for example , can be a fixed connection, or a detachable connection, or integrated; it can be a mechanical connection, or an electrical connection; it can be a direct connection, or an indirect connection through an intermediary; it can be the internal communication or connection between two components. As for the interaction relationship between two elements, unless otherwise clearly defined, those skilled in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

Example:

The present invention provides a kiwi fruit endophytic antagonistic fungus Fusarium nodosa varietal and its application, as shown in Figure 1-25, Fusarium nodosa varietal strain (J-1) of the present invention has been produced in It was deposited in the China Center for Type Culture Collection (Address: Wuhan, China, Wuhan University, Zip Code: 430072, Tel: 027-68754052) on May 11, 2021, deposit number: CCTCC NO: M2021510.

1. Separation and purification of kiwifruit antagonistic endophytic fungi: kiwifruit fruits were collected from the red-heart kiwifruit production area of Shuicheng County, Liupanshui City, Guizhou Province, rinsed with sterile water, dried and stored at 4°C, and the surface of the fruit was sterilized with 75% alcohol and rinsed with sterile water After washing 3 times, inoculate kiwifruit tissue pieces on potato agar medium (PDA) and culture them in a constant temperature incubator at 28°C. After obvious hyphae grow on the edge of the tissue, take the tip hyphae for further purification and culture, and purify 2-3 times , and the obtained strains were stored in a -80°C low-temperature refrigerator for later use.

2. Screening of endogenous antagonistic fungi: inoculate Alternaria bacterium in the center of PDA medium by plate confrontation method, and inoculate 4 cakes of antagonistic bacteria to be screened at 2cm around the pathogenic bacteria in cross symmetry, and keep the temperature at 28°C After dark culture in the box for 7 days, the growth of Alternaria mycelium was observed, and the antagonistic strain J-1, which can significantly inhibit the growth of Alternaria mycelium, was obtained.

3. Identification of morphological characteristics of strain J-1: strain J-1 grows slowly on PDA medium, and the colonies of strain J-1 are irregular and circular after being cultured at 28°C for 15 days, with a growth rate of 2.013mm/d. Form a yeast-like slimy layer, as shown in Figure 1, with few aerial hyphae and dense mycelium, spread on the medium; the middle part of the colony is purple-red after about 15 days of cultivation, and the color gradually becomes lighter from the center of the colony to the edge , the edge is off-white, and the middle mycelium is an upright tree crown (accompanying drawing 2, accompanying drawing 3).

J-1 bacterial strain can form myxospore mass in the middle part of bacterial colony after cultivating about 30d on the PDA medium, (accompanying drawing 4), translucent, sporulation cell single bottle stem (accompanying drawing 5), contains a large amount of conidia, Conidia are colorless, and there are many large conidia with 1 to 3 septa, mostly 3 septa, and very few small conidia with 1 septum. The spores are sickle-shaped, with slightly blunt ends. Sometimes it is beak-shaped, with a size of (29-46.86) μm×(3.8-5.9) μm (Fig. 6 and Fig. 7).

Under the microscope, the hyphae are slender, compact, without septa, and have many branches (accompanying drawing 8, accompanying drawing 9).

According to the morphological characteristics of the antagonistic fungus, it belongs to the genus Fusarium (Fusarium spp.).

4. J-1 Molecular Biological Identification: Genomic DNA of the strain was extracted, using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and Btu-F-F01 (5'-CAGACCGGTCAGTGCGTAA- 3'), Btu-F-R01(5'-TTGGGGTCGAACATCTGCT-3') was used to amplify the antagonized fungal internal ribosome transcribed spacer and β-tubulin gene by PCR, and the obtained length was 545bp (ITS) and 918 (β- tub) gene fragment (see the sequence list for details), the obtained sequence was uploaded to the NCBI website (https://www.ncbi.nlm.nih.gov) for BLAST comparison, and its ITS sequence and β-tub sequence were consistent with the section Fusarium meridmoides var. violaceum was the most similar, with homology of 99.63% and 100%, respectively. The multigene system was constructed by using the maximum parsimony method (MP) of PAUP software combined with antagonistic bacteria ITS and β-tub sequences In the developmental tree (accompanying drawing 10), the J-1 strain and Fusarium meridmoides var. violaceum are clustered into one branch, and the support rate is 100%, so it is clear that the J-1 strain is Fusarium meridmoides var. violaceum.

5. The growth characteristics of the J-1 strain on different media: After the J-1 strain was cultured on PDA, PSA, OA, CA, Czapek, and Bilais media, its growth characteristics were significantly different, and it grew slowly on PDA media , the mycelia in the early stage of growth are off-white to orange-yellow, forming a yeast-like sticky slippery layer, producing tree-crown mycelia in the center of the colony, and the middle part of the colony is purple (accompanying drawing 11); the mycelium grows relatively vigorously on the PSA medium, and the colony Purple red, and the color is further deepened (accompanying drawing 12); On the OA medium, there are few hyphae, and the hyphae are relatively sparse and mostly spread on the medium, and the edge hyphae are yellow to orange in the later stage of culture (accompanying drawing 13) On the CA substratum, the mycelium is tiled on the substratum, only a small amount of mycelium in the middle is purple, and most of the mycelium is white (accompanying drawing 14); Growth is slow on the Czapek substratum, and the mycelium amount is few, is White to light yellow (accompanying drawing 15); On Bilais culture medium, mycelia growth amount is very little, white, does not produce purple pigment (accompanying drawing 16).

6. The broad-spectrum antagonistic effect of the antagonistic fungi of the J-1 strain: Inoculate Alternaria, Phomopsis, Botrytis, and Cocoa spp. in the center of the PDA medium using the plate confrontation method, and the cross is symmetrical Inoculate 4 bacterial cakes of J-1 strain at 2 cm around the pathogenic bacteria, and culture them in the dark in an incubator at 28°C for 7 days to observe the growth of the pathogenic bacteria. Calculate the antagonistic index according to the following formula: antagonistic index = (radius of inhibition zone - radius of antagonistic bacteria) / radius of inhibition zone; where: radius of inhibition zone = distance from the center of the colony of antagonistic bacteria to the edge of the hyphae of the pathogenic bacteria, and the J-1 strain was found It has obvious antagonistic effect on various pathogenic bacteria, and the antagonistic indices are 48.98% (accompanying drawing 17), 43.9% (accompanying drawing 18), 27.12% (accompanying drawing 19) and 23.26% (accompanying drawing 20).

7. Identification of antagonistic endophytic fungus J-1

Observation of morphological characteristics of antagonistic endophytes: (1) Morphological characteristics: Inoculate J-1 strains on six mediums of PDA, PSA, OA, CA, Czapek, and Bilais respectively, and observe the morphology and color of mycelia after culturing at 28°C for several days characteristics such as changes and growth rates. (2) Observation of microcosmic morphology: Take 3-5 mm ² sterile carnation leaves, put them on the surface of sterilized WA medium cooled to 45°C, inoculate the J-1 bacterial block near the carnation leaves, and cultivate them for 15 days to promote their growth. Sporulation. After sporulation, observe its mycelial morphology and sporulation structure under an optical microscope, take an appropriate amount of culture with an inoculation needle, place it on a glass slide with a drop of sterile water, cover it with a cover glass, observe the spore morphology, and measure the spores size.

8. The inhibitory effect of J-1 strain fermentation broth on Alternaria:

Take 3 J-1 antagonistic bacteria cakes and inoculate them in 100ml PDB medium, culture them on a constant temperature shaker at 28°C and 120rpm for 7 days, filter the fermentation broth with a 0.22μm filter membrane, and set aside for later use. Taking Alternaria alternata as the target bacteria, inoculate it in the center of PDA medium containing 20% sterile filtrate, culture it in the dark at 28°C for 7 days, and observe the growth of pathogenic bacteria.

J-1 sterile fermentation filtrate has significant inhibitory effect on Alternaria, with an average inhibition rate of 69.93% (accompanying drawing 21).

9. Sensitivity of J-1 strain fermentation broth to temperature:

Take 3 J-1 bacterial blocks with a 6mm sterile puncher, put them into a conical flask containing 100ml of PDB culture solution, culture at 28°C, 150r/min for 7 days, and then use 8 layers of sterile gauze to remove the hyphae to obtain the filtrate. After the filtrate was sterilized by 0.22μm filter membrane, it was placed at -80°C, -20°C, 0°C, 20°C, 80°C, and 120°C for 30 minutes, and it was equilibrated at room temperature for 3 minutes, and it was mixed with PDA respectively to make For a PDA plate with a filtrate content of 20%, take the PDA plate without sterile filtrate as a control, inoculate Alternaria in the center of the plate, repeat each treatment 3 times, place it in an incubator at 28°C for 7 days, and calculate the inhibitory effect: Inhibition rate=[(control colony diameter-treated colony diameter)/control colony diameter]×100%.

After the sterile filtrate of J-1 strain was treated at -80°C, -20°C, 0°C, 20°C, 80°C, and 120°C for 30 minutes, there was no significant difference in the inhibitory activity against Alternaria compared with the untreated control , showing that the main antibacterial active ingredient produced by the antagonistic fungal strain is not sensitive to ambient temperature, and its activity has no significant change after high temperature and low temperature treatment (accompanying drawing 22).

10. The acid-base stability of J-1 strain fermentation broth:

Use 2mol/L NaOH or 2mol/L HCl to adjust the pH of J-1 sterile fermentation filtrate to 3, 4., 5, 6, 7, 8, 9, 10, 11 respectively, and adjust the pH of all treatments after standing for 1h. It is 7, it is mixed with PDA respectively to make the PDA plate that filtrate content is 20%, is contrast with the PDA plate that does not contain sterile filtrate, inoculates Alternaria in the center of each plate, every treatment is repeated 3 times, placed in Cultivate in an incubator at 28°C for 7 days, and calculate the inhibitory effect: inhibition rate=[(control colony diameter-treated colony diameter)/control colony diameter]×100%.

Adjust the pH of the sterile filtrate of the J-1 strain to 3, 4, 5, 6, 7, 8, 9, 10, and 11, and then adjust the pH to 7 in sequence after standing for 1 hour. The sterile filtrate is still effective against Alternaria It has strong antibacterial activity, indicating that acid-base environmental conditions have no significant impact on the main antibacterial active ingredients produced by the antagonistic fungal strain (accompanying drawing 23).

12. Effect of storage time on the activity of J-1 strain fermentation broth:

The J-1 sterile fermentation filtrate obtained from the culture was stored at room temperature for 0d, 15d, 30d and 45d, and the sterile filtrate at different storage time points was mixed with PDA to make a PDA plate with a filtrate content of 20%. The PDA plate was used as a control, and Alternaria was inoculated in the center of each plate, and each treatment was repeated 3 times, placed in a 28°C incubator and cultured for 7 days, and the inhibition effect was calculated: inhibition rate = [(control colony diameter - treatment colony diameter)/ Control colony diameter] × 100%.

After placing the sterile filtrate of the same batch of J-1 bacterial strains at room temperature for 0d, 15d, 30d and 45d, there was no significant change in its inhibitory effect on Alternaria, indicating that the antibacterial active substances produced by the antagonistic strains last longer ( Accompanying drawing 24).

13. Indoor biocontrol effect of fermentation broth of J-1 strain:

Select Hongyang kiwi fruit with uniform size and color, no mechanical damage, diseases and insect pests, soak and disinfect in 2% sodium hypochlorite for 2 minutes, rinse with clean water and dry naturally. Using the same hole injury inoculation method, use a 5mm sterile puncher to drill 4 holes (4mm wide, 5mm deep) at the equator of the fruit. Inoculate 4 wells respectively: T: 10 μl Alternaria spore suspension (concentration: 10 ⁶ ml ^-1 ) + 10 μl fermentation broth; W-1: 10 μl Alternaria spore suspension (concentration: 10 ⁶ ml ^{-1 ) 1} ); J-1: 10 μl fermented liquid; CK: 10 μl sterile water, (in all treatments, J-1 fermented liquid was not sterilized by a 0.22 μm filter membrane) after the treatment, the kiwi fruit was placed in 25°C constant temperature and moisture-retaining culture, and observed The inhibitory effect of fermentation broth on Alternaria and whether it is pathogenic to kiwifruit.

The results showed that the J-1 fermentation broth could significantly inhibit the infection of Alternaria and reduce the infection speed of Alternaria. Meanwhile, its fermentation broth had no pathogenicity to Hongyang kiwifruit (accompanying drawing 25).

An endophytic fungus isolated from kiwifruit that has a strong antagonistic effect on various pathogenic bacteria such as Alternaria sp. Variant (Fusarium meridmoides var. violaceum), the aseptic fermentation filtrate of this strain has strong temperature adaptability and acid-base adaptability, and its antibacterial active substances have a long lasting effect. After the kiwi fruit is treated with the antagonistic bacteria fermentation liquid, it can effectively inhibit the infection of Alternaria spp., and the fermentation liquid has no pathogenicity to kiwi fruit, so it has potential commercial development and application value, and has a wide range of applications in the biological control of kiwi fruit rot disease. application prospects.

The parts of the present invention that are not described in detail are known technologies of those skilled in the art.

The embodiments of the present invention have been presented for purposes of illustration and description, but are not intended to be exhaustive or to limit the invention to the form disclosed. Many modifications and changes will be apparent to those of ordinary skill in the art. The embodiment was chosen and described in order to better explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention and design various embodiments with various modifications as are suited to the particular use.

<110> Guizhou University

<120> An endophytic antagonistic fungus Fusarium nodosa varietal in kiwifruit and its application

<160> 2

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<213> Fusarium merismoides var. violaceum

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<212>DNA

<213> Fusarium meridmoides var. violaceum

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Claims (3)

1. A kiwi fruit endophytic antagonistic fungus Fusarium meridmoides var. The number is: CCTCC NO: M2021510, and the date of deposit is: May 11, 2021. 2. the purposes of a kiwifruit endophytic antagonistic fungus Fusarium nodosa Viola varietal strain in antagonizing kiwifruit fruit rot pathogenic bacteria as claimed in claim 1, characterized in that: said kiwifruit fruit rot pathogenic bacteria Alternaria, Phomopsis and Botrytis. 3. the application method of the kiwi fruit endogenous antagonistic fungus Fusarium nodosa Viola varietal bacterial strain according to claim 2, is characterized in that: described method is: bacterial strain J-1 is inoculated in kiwi fruit fruit after PDA medium culture superior.

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