Inoculation method for colonizing new host plant by endophytic fungi

Abstract

The invention belongs to the technical field of plant fungus inoculation, and particularly relates to an inoculation method for colonizing a new host plant through endophytic fungi. The method specifically comprises the following steps: S1, inoculating endophytic fungi into a PDA culture medium, and culturing in a mould incubator for 24-26 days to obtain endophytic fungi hyphae; s2, placing the disinfected seeds in a water agar culture medium, and accelerating germination in an incubator to obtain plant aseptic seedlings; s3, when the aseptic seedlings of the plants grow to 3-5cm, scratching the aseptic seedlings of the plants, stuffing endophytic fungus hyphae into wounds, continuously placing the aseptic seedlings of the plants in the water agar culture medium, and culturing the aseptic seedlings of the plants in an incubator; and S4, planting the plant aseptic seedlings cultured for 3-7 days in the step S3 into aseptic soil, and observing whether hyphae exist in the plants or not after one month. In the technical scheme provided by the invention, endophytic fungi which cannot infect roots can use the method provided by the invention; efficiency and success rate are high.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fungus inoculation, and in particular relates to an inoculation method for endophytic fungi to colonize new host plants. Background Art

[0002] The invasion and colonization of endophytic fungi in plant tissues is a prerequisite for their function. ) and locoweed endophytes, which can naturally infect endophytic fungi and produce a certain proportion of fungus-carrying plants and seeds through vertical transmission. Most of the other endophytic fungi are mainly inoculated artificially, mostly using the bacterial cake inoculation method and bacterial liquid root irrigation method.

[0003] The mushroom cake inoculation method can directly introduce endophytic fungi into the rhizosphere of plants, achieving a more natural colonization process; for some plants with strong rooting ability, the mushroom cake inoculation method can better achieve the colonization of endophytic fungi.

[0004] However, for some difficult-to-root plants or seedlings, the mushroom cake may not be easy to attach or absorb, and the colonization effect is limited; during the preparation of the mushroom cake, the survival rate and activity of endophytic fungi may be affected, thereby affecting the transfer effect; the preparation and preservation conditions of the mushroom cake are relatively harsh, requiring professional equipment and technical support.

[0005] The root irrigation method can directly inject the endophytic fungus culture solution around the plant roots to simulate the natural colonization process; it is suitable for plants at different growth stages, including seedlings and mature plants. However, the stability of the bacterial solution is poor and it needs to be irrigated in time, otherwise the activity of the strain may decrease; the contamination rate of the bacterial solution directly poured into the soil is extremely high; for plants with poorly developed or loose root systems, the irrigation effect is not ideal.

[0006] Most existing methods are only applicable to endophytic fungi that can infect roots, and have high requirements on the strain types. As an endophytic fungus, it cannot invade the plant from the roots, and it cannot infect intact plants without any wounds.

[0007] Therefore, for As endophytic fungi, it is of great significance to develop a new inoculation method for endophytic fungi to colonize new host plants. Summary of the invention

[0008] In order to solve the above technical problems, the present invention discloses an inoculation method for endophytic fungi to colonize new host plants, the specific steps of which are:

[0009] S1. Inoculate the endophytic fungus into the PDA medium and place it in a fungus incubator for 24 to 26 days to obtain endophytic fungal hyphae;

[0010] S2. Placing the sterilized seeds on a water agar medium and germinating them in an incubator to obtain sterile seedlings;

[0011] S3. When the sterile plant seedlings grow to 3-5 cm, scratch the sterile plant seedlings, insert the endophytic fungal hyphae into the wound, and then continue to place them in a water agar medium and culture them in an incubator;

[0012] S4. Plant the sterile seedlings of plants cultured in S3 for 3 to 7 days in sterile soil and observe the presence of hyphae after 1 month.

[0013] In S1, the preparation method of the PDA culture medium is: dissolving PDA in distilled water, sterilizing at 110-130° C. for 10-20 min, to obtain the PDA culture medium; the temperature in the mold incubator is 24-26° C.;

[0014] Preferably, in S1, the concentration of the PDA culture medium is 0.03-0.06 g / ml; further preferably, the concentration of the PDA culture medium is 0.046 g / ml.

[0015] In S2, the seed disinfection process is specifically as follows: the seeds are rinsed with tap water for 3 times, then soaked in sterile water for 12 hours, then surface disinfected with sodium hypochlorite solution for 3 minutes, rinsed with sterile water for 4 times, and finally disinfected with alcohol for 4 minutes and rinsed with sterile water for 4 times;

[0016] Wherein, the concentration of the sodium hypochlorite solution is 2.5%, and the concentration of the alcohol is 75%.

[0017] In S2, the concentration of the water agar medium is 0.01 to 0.013 g / mL; the temperature of the incubator is 25 to 30° C.;

[0018] Preferably, in S2, the concentration of the water agar medium is 0.012 g / mL; and the temperature of the incubator is 28°C.

[0019] The specific steps in S3 are: when the sterile seedlings of the plant grow to 3 to 5 cm, place the sterile seedlings flat on the lid of the culture dish, fix the roots with sterilized tweezers, and use a sterilized scalpel to scratch the sterile seedlings of the plant at a distance of 2 to 3 mm from the roots where the white and green stripes are interlaced. At this time, the sterile seedlings of the plant cannot be scratched, and then the endophytic fungal hyphae are stuffed into the wound and continued to be placed in a water agar medium and cultured in an incubator.

[0020] In S4, the sterile soil is obtained by high temperature and high pressure sterilization at 120-125°C;

[0021] Preferably, in S4, the sterile soil is obtained by sterilization at a high temperature and high pressure at 121°C.

[0022] The application of the above inoculation method in colonizing endophytic fungi is also within the scope of protection of the present invention.

[0023] The endophytic fungi are endophytic fungi that cannot infect from the roots of plants or cannot infect from intact plants without wounds.

[0024] Specifically, in some embodiments of the present invention, the endophytic fungi include but are not limited to those described in the embodiments. It is an endophytic fungus.

[0025] Specifically, in some embodiments of the present invention, through the above-mentioned inoculation method, the researchers of the present invention will Endophytic fungi were transferred to 300 Buton barleys or 350 Sudan grasses; after observation, the success rate of transferring Buton barley was as high as 70% (homologous), and the success rate of transferring Sudan grass was 7% (different sources), which proved the application prospect of the above method of the present invention in the field of inoculating endophytic fungi that cannot be infected from the roots.

[0026] Specifically, in some embodiments of the present invention, DNA identification and molecular identification were performed on the strains obtained by the above-mentioned inoculation method, proving that the inoculation of endophytic fungi was successful, and a new endophytic fungus-Boughton barley symbiont was obtained.

[0027] Beneficial effects: In the technical solution provided by the present invention, the hardness of the water agar culture medium is appropriate; the sterile seedling incision method is used to transfer endophytic fungi to a new host, and the roots (seeds) are fixed with tweezers, which will not affect the growth of the plant; in addition, the method provided by the present invention can be used for endophytic fungi that cannot infect the roots; the efficiency is high and the success rate is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0029] Figure 1 Flow chart of the inoculation method in an embodiment of the present invention;

[0030] Figure 2 It is a phylogenetic tree constructed based on tef sequence homology in the embodiment of the present invention;

[0031] Figure 3 It is a phylogenetic tree constructed based on tub sequence homology in the embodiment of the present invention;

[0032] Figure 4 It is a diagram of the comparison results of tef and tub genes in an embodiment of the present invention, wherein A is the comparison result of the new symbiont ~tef sequence and W1~tef, and B is the comparison result of the new symbiont ~tub sequence and W1~tub. DETAILED DESCRIPTION

[0033] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0034] Embodiment 1:

[0035] (1) Prepare PDA medium (purchased from Hangzhou Baisi Biotechnology Co., Ltd.) according to the following formula: weigh 46.0 g of PDA, add 1000 mL of distilled water, stir and heat to boil until completely dissolved, and sterilize at 121°C for 15 min to obtain PDA medium;

[0036] (2) The endophytic fungi were inoculated on PDA medium and cultured in a fungus incubator at 25°C for 25 days;

[0037] (3) 12 g agar per 1000 mL distilled water (COOLABER SCIENCE &

[0038] TECHNILOGY) Prepare water agar medium;

[0039] (4) Preparation of sterile seedlings: Rinse the seeds with tap water three times, then soak them in sterile water for 12 hours, then disinfect them with 2.5% sodium hypochlorite solution for 3 minutes, rinse them with sterile water four times, disinfect them with 75% alcohol for 4 minutes, and rinse them with sterile water four times. Place the disinfected seeds on water agar medium and germinate them in an incubator at 28°C;

[0040] (5) When the seeds grow to 3-5 cm, inoculate: Place the sterile seedlings flat on the lid of the culture dish, fix the roots (seeds) with sterilized tweezers, and use a sterilized scalpel to scratch the white and green area 2-3 mm away from the roots (do not scratch through). Then use the sterile scalpel to scratch a small amount of endophytic fungal hyphae (after growing on the PDA plate for 25 days), insert it into the wound, and then place it on the water agar medium. After placing it in the incubator for 3-7 days, plant it in sterile soil sterilized at 121℃. Water the nutrient solution according to the growth status of different plants. One month later, use an optical microscope to observe whether there is mycelium in the plants.

[0041] Evaluation of vaccination effect:

[0042] According to the above inoculation steps, researchers of the present invention can inoculate The endophytic fungi were transferred to 300 Buton barley or 350 Sudan grass; among them, the success rate of transferring Buton barley was as high as 70% (homologous), and the success rate of transferring Sudan grass was 7% (different origin).

[0043] Take the example of the inoculation of endophytic fungi of Wensu Buton barley into Gansu Buton barley plants:

[0044] (1) Identification of purified strain DNA:

[0045] DNA of the strain was extracted and purified, and amplified with primers of gene tef A and tub B. The amplified products were subjected to electrophoresis, and the recovered fragments were connected to the PMD18-T vector and transferred into Escherichia coli DH5α. Three to five positive clones were selected for sequencing for each sample. The sequencing results were aligned using Blast n at NCBI, and representative sequences of endophytic fungi with high similarity were downloaded. The sequences were processed using MEGA 10.1.7 and the neighbor-joining method (NJ) was used to establish a phylogenetic tree. The optimal model parameters were input, and 1000 random repeated samplings were used.

[0046] The strain was amplified with the transcription elongation factor tef primer to obtain a 805bp gene sequence. The sequence comparison analysis showed that the tef sequence of the strain was similar to that of Neotyphodium sp. and bromicola, the TEF sequences were 99.87% and 98.32% respectively. The phylogenetic tree constructed based on the TEF sequence homology showed that the strain was related to Neotyphodium sp. and Bromicola gathered on a large branch ( Figure 2 In addition, DNA sequence amplification was performed using tubulin tub as primer to obtain a 922 bp gene sequence. Sequence comparison analysis results showed that the DNA sequence of the strain was consistent with that of E. bromicola, KP877325, KX219728, and KP877325, respectively.

[0047] sp.DNA sequence homology is 99.88% and 99.78% respectively. The phylogenetic tree constructed based on tub sequence homology is shown in Figure 3 As shown, the strains were different from E. bromicola, sp. clustered on one branch. Therefore, this strain was identified as bromicola.

[0048] (2) Molecular identification of endophytic fungi in new hosts

[0049] Fungal DNA was extracted using a fungal DNA extraction kit, and the purified fungal DNA was used as a template to amplify two pairs of endophytic fungal housekeeping genes, tef and tub, using a PCR instrument for molecular identification. The PCR amplification results were detected by agarose gel electrophoresis and sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing.

[0050] The cloned tef and tub genes were sent for sequencing, and the sequencing results were compared with the sequences of the inoculated endophytic fungi sequenced by our research group. The results showed that both tef and tub genes were successfully aligned ( Figure 4 ), proving that the access to endophytic fungi was successful, and a new endophytic fungus-Buton barley symbiosis was obtained, which can be used for subsequent experiments, Figure 4 In the figure, W1 is the original inoculation sequence; (A): the alignment result of the new symbiont ~tef sequence and W1~tef; (B): the new symbiont

[0051] Alignment results of ~tub sequence and W1~tub.

[0052] The present invention provides a method for inoculating an endophytic fungus to colonize a new host plant. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for inoculating a new host plant with an endophytic fungus, characterized in that: The specific steps are: S1. Inoculate the endophytic fungus into the PDA medium and place it in a fungus incubator for 24 to 26 days to obtain endophytic fungal hyphae; S2. Placing the sterilized seeds on a water agar medium and germinating them in an incubator to obtain sterile seedlings; S3. When the sterile plant seedlings grow to 3-5 cm, scratch the sterile plant seedlings, insert the endophytic fungal hyphae into the wound, and then continue to place them in a water agar medium and culture them in an incubator; S4. Plant the sterile seedlings of plants cultured in S3 for 3 to 7 days in sterile soil and observe the presence of hyphae after 1 month.

2. The inoculation method according to claim 1, characterized in that In S1, the preparation method of the PDA culture medium is: dissolving PDA in distilled water, sterilizing at 110-130° C. for 10-20 min, to obtain the PDA culture medium; the temperature in the mold incubator is 24-26° C.

3. The inoculation method according to claim 1 or 2, characterized in that: The concentration of the PDA culture medium is 0.03-0.06 g / ml.

4. The inoculation method according to claim 1, characterized in that In S2, the seed disinfection process is specifically as follows: rinse the seeds with tap water for 3 times, then soak them in sterile water for 12 hours, then disinfect the surface with sodium hypochlorite solution for 3 minutes, rinse with sterile water for 4 times, and finally disinfect with alcohol for 4 minutes and rinse with sterile water for 4 times.

5. The inoculation method according to claim 4, characterized in that The concentration of the sodium hypochlorite solution is 2.5%, and the concentration of the alcohol is 75%.

6. The inoculation method according to claim 1, characterized in that: In S2, the concentration of the water agar culture medium is 0.01-0.013 g / mL; and the temperature of the incubator is 25-30°C.

7. The inoculation method according to claim 1, characterized in that: The specific steps of S3 are: when the sterile seedlings of the plant grow to 3 to 5 cm, place the sterile seedlings flat on the lid of the culture dish, fix the roots with sterilized tweezers, and use a sterilized scalpel to scratch the sterile seedlings of the plant at a distance of 2 to 3 mm from the roots where the white and green are alternating. At this time, the sterile seedlings of the plant cannot be scratched, and then the endophytic fungal hyphae are stuffed into the wound and continued to be placed in a water agar culture medium and cultured in an incubator.

8. The inoculation method according to claim 1, characterized in that: In S4, the sterile soil is obtained by high temperature and high pressure sterilization at 120-125°C.

9. Use of the inoculation method according to any one of claims 1 to 8 in colonizing endophytic fungi.

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