WO2019062354A1

WO2019062354A1 - Fungal elicitor, preparation method therefor, and method for rapid propagation of bletilla striata seedlings using fungal elicitor

Info

Publication number: WO2019062354A1
Application number: PCT/CN2018/100273
Authority: WO
Inventors: 席刚俊; 杨鹤同; 史俊; 张靓; 郑凯
Original assignee: 江苏农林职业技术学院
Priority date: 2017-09-26
Filing date: 2018-08-13
Publication date: 2019-04-04
Also published as: CN107629966A

Abstract

Provided are a fungal elicitor, a preparation method therefor, and a method for the rapid propagation of Bletilla striata seedlings using the fungal elicitor. The fungal elicitor is prepared by Purpureocillium lilacinum JSNLBJ-001 with the deposit number CGMCC NO. 13690. In the aseptic seeding stage of Bletilla striata seeds, by adding a suitable concentration of the fungal elicitor, in place of other artificial hormones, to a germination medium of Bletilla striata seeds, and aseptically seeding Bletilla striata seeds in the medium, the method can realize one-step seedling formation, shorten the production cycle of Bletilla striata tissue culture seedlings, and save on production costs compared with the original production process.

Description

Fungus elicitor, preparation method thereof and method for rapid propagation of white peony seedling by using the fungus elicitor

Technical field

The invention relates to the field of plant tissue culture and microbial fermentation, in particular to a fungal elicitor, a preparation method thereof and a method for rapid propagation of white peony seedlings by using the fungus elicitor.

Background technique

Under natural conditions, orchids must establish a symbiotic relationship with mycorrhizal fungi to complete their normal growth and development. In the mutually beneficial symbiosis system, the mycorrhizal fungi can provide the host plants with the vitamins and hormones necessary for growth and development. Fungal elicitors are derived from microbial molecules and are a class of substances that cause plant cell synthesis to accumulate secondary metabolite activity. Fungal elicitors include mycelium, fungal culture filtrate, fungal spores, homogenates, and fungal cell wall components. As a "vaccine" of plants, fungal elicitors can also be recognized as signalling substances by plants at very low concentrations ( ^10-9 or lower), inducing the plant's own immune system, and ultimately giving plants the ability to resist disease.

At present, the application of fungal elicitors to the production of seedlings in white peony has not been reported.

Summary of the invention

OBJECT OF THE INVENTION: In order to solve the problems in the prior art, one of the objects of the present invention is to provide a fungal elicitor which can be applied to the rapid propagation of white peony seedlings, and the second object of the present invention is to provide the fungal elicitor. The preparation method, the third object of the present invention, is to provide a medium containing the fungal elicitor, and the fourth object of the present invention is to provide a method for rapid propagation of white peony seedlings using the fungal elicitor.

Technical Solution: The method for preparing a fungal elicitor according to the present invention is prepared by a Lactobacillus brevis which is named Purpureocillium lilacinum JSNLBJ-001, and the preservation number is CGMCC NO: 13690.

The P. glabrata according to the present invention is named as Purpureocillium lilacinum JSNLBJ-001, and is classified as Purpureocillium lilacinum, and its taxonomic status is Ascomycetes. The genus Pleurotus sinensis, strain No. JSNLBJ-001, has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee. The deposit number is CGMCC NO: 13690, and the deposit date is March 10, 2017. The date of deposit is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101.

The above strain was cultured on PDA medium for 7 days, the colony diameter was 3.5 cm, and the growth was slow. The colony is dark green, felt-like, leathery, with neat edges and a transparent outer ring of about 1 mm.

Specifically, the method for preparing the fungal elicitor comprises: inoculating the P. glabrata in a medium for fermentation culture, and sterilizing the fermentation broth and the hyphae after the fermentation is completed.

Before inoculation, the P. glabrata was inoculated on a PDA plate for a period of time, and then the cake was inoculated into a medium for fermentation culture.

In the fermentation culture, the medium is a PDA liquid medium.

The fermentation culture is a shaking culture at a rotation speed of 120 to 150 r/min, and the fermentation culture temperature is 25 to 27 ° C for 10 to 15 days.

The present invention also provides a fungal elicitor prepared according to the above preparation method.

The present invention further provides a medium for rapid propagation of white peony seedlings comprising the fungal elicitor.

In the medium for rapid propagation of the white peony seedling, the concentration of the fungal elicitor is 50 to 200 ml/L, preferably 80 to 100 ml/L.

Specifically, the culture medium of the white peony seedling rapid propagation comprises: N6 basic medium + the fungal elicitor + Huabao No. 1 4~10g/L+25-30g/L sucrose +5-6.5g / L agar + 50 ~ 80g / L potatoes + 80 ~ 100g / L banana.

Huabao No. 1 is produced by Hyponex, and the product number is Hyponex NO.1.

Potatoes and bananas are peeled and then pulverized into a mud with a cooking machine. Huabao No. 1 mainly provides the necessary N, P, K and other elements and related trace elements for the seed germination and growth of white peony;

Potatoes are rich in starch, which can provide carbon source for germination and growth of white peony seeds. In addition, potatoes contain certain phytohormones, which can promote the germination of white peony seeds. The potatoes also contain certain peroxidase and superoxide radicals. Enzymes and vitamin C can prevent excessive oxygen in the air, causing oxygen negative ions to cause damage to tissue culture plants;

Bananas contain a large amount of sugars, which provide a carbon source for growth, and plant hormones such as cytokinins, auxins, ethylene and gibberellin in bananas can promote the germination and growth of white peony seeds.

Finally, the present invention provides a method for rapid propagation of white peony seedlings using a fungal elicitor, comprising: cultivating a white peony seed in a medium containing a fungal elicitor to obtain a tissue culture seedling.

The medium may be a medium for rapid propagation of the above-mentioned white peony seedlings.

Prior to sowing, the white peony seeds were sterilized.

In the rapid propagation of white peony seedlings, the culture conditions are: temperature 25±5°C, illumination 1500～2500Lx, illumination time 8～12h/d, culture time 100～120 days.

After sowing of white peony seeds, during the cultivation process, the first 30 to 50 days after sowing, the temperature is controlled at 20-22 ° C, the illumination is controlled at 1500-1800 LX, and the illumination time is controlled at 8-10 h/d. The seeds are germinated to a height of 2 cm. After the seedlings, the temperature is adjusted to 22-28 ° C, the light is adjusted to 1800-2500 LX, and the illumination time is adjusted to 10-12 h/d for 50-90 days to obtain transplantable finished tissue culture seedlings.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention prepares a fungal elicitor by using the genus Bacillus licheniformis deposited under the accession number CGMCC No. 13690, and germinated in the white peony seed during the aseptic seeding stage of the white peony seed. Adding a suitable concentration of fungal elicitor to the culture medium instead of other artificial hormones, aseptically sowing the white peony seeds in the above medium, can form a seedling at a time, compare the original production process, can greatly shorten the production cycle of the white peony seedlings, and save a lot of The production cost has broad application prospects.

Detailed ways

The present invention will be further clarified by the following specific examples, which are to be construed as illustrative only and not to limit the scope of the invention. Modifications are within the scope defined by the claims appended hereto.

Isolation and identification of the strain of Example 1

The strain JSNLBJ-001 was isolated in May 2015 in a plant with good growth conditions in the Baiji planting area of Jiangsu Maoshan Daodi Medicine Co., Ltd., Jurong City, Jiangsu Province.

The separation method is: taking wild fresh white radix root, rinsing the substrate soil on the surface of the root section with running tap water, and then using a filter paper to absorb the surface moisture in the ultra-clean workbench. Rinse with sterile deionized water, then immerse in 75% alcohol for 45 s, and soak in 2% sodium hypochlorite solution for 5 min. After disinfection is completed, rinse with sterile deionized water for 5-6 times to completely remove sodium hypochlorite, use the sterile filter paper to absorb the residual water droplets on the root surface, and then cut the root section to 0.5mm with a sterile scalpel. Organization segment. The tissue segments were transferred to PDA medium plates supplemented with streptomycin sulfate (100 μg/mL), and each plate was inoculated with 5 tissue sections, each of which was subjected to 15 plates. The endophytic fungus obtained by the isolation is subjected to a back-test to obtain the strain of the present invention, and the strain is named as JSNLBJ-001, which can obviously promote the growth of the seedlings of the white peony seedlings and increase the survival rate of the seedlings.

The colony characteristics of strain JSNLBJ-001 are as follows:

Morphological observation showed that the culture was slow on the PDA medium for 7 days, the colony diameter was 3.5 cm. The colony is dark green, felt-like, leathery, with neat edges and a transparent outer ring of about 1 mm.

The entire sequence of the ITS region was amplified and sequenced, and the full sequence of the 16s RNA obtained by PCR amplification was as follows. The full sequence obtained by PCR amplification (SEQ ID NO. 1):

The morphological and molecular biological identification indicated that the strain was Purpureocillium lilacinum, and its taxonomic status was ascomycetes of the genus Aspergillus, the genus Aspergillus, and the strain number was JSNLBJ. -001, has been deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, the deposit number is CGMCC NO: 13690, and the deposit date is March 10, 2017. The deposit address is No. 1 Courtyard of Beichen West Road, Chaoyang District, Beijing. No. 100101, Institute of Microbiology, Chinese Academy of Sciences.

Example 2

A method for rapid propagation of white peony seedlings using a fungal elicitor, comprising the following steps:

(1) Preparation of fungal elicitor: P. glabrata JSNLBJ-001 was inoculated on PDA plate medium for 10 days, then punched with a puncher to make a cake, and the cake was inoculated into pre-packaged and sterilized. In a good PDA liquid medium, shake culture on a shaker (120r/min, 27 °C) for 12 days. After the fungus is fully fermented, the fermentation broth and the hyphae ball are pulverized into a uniform mixture by a stirrer at 121 ° C. Sterilize for 20 minutes, cool and store in a refrigerator at 4 °C for use.

(2) The seedling rapid propagation medium was configured according to the following components: the fungal elicitor in step (1) 50 ml/L+N6 basal medium + Huabao 1 8 g/L + sucrose 30 g/L + agar 6 g/ L+ potato 50g/L+ banana 80g/L, sterilized at 121 ° C for 20 min for use. Potatoes and bananas are peeled and then pulverized into a mud with a cooking machine.

(3) Seeding of white peony seeds for cultivation and production of seedlings: The sorghum fruit is sterilized by 70% alcohol, 0.1% liters of mercury and sterile water in sequence, and the sorghum seeds are cut out under aseptic conditions, and the seeds are evenly spread. In the medium of step (2), after culturing for 40 days in a culture chamber at a temperature of 22 ° C, a light of 1500 Lx, and an illumination time of 8 h/d, after the seed is germinated into a seedling of 2 cm height, the temperature is adjusted to 28 ° C, and the light is adjusted to 2500 LX. The illumination time was adjusted to 12h/d for 80 days to obtain transplantable finished tissue culture seedlings.

Example 3

(1) Preparation of fungal elicitor: B. glabrata JSNLBJ-001 was inoculated on PDA plate medium for 8 days, then punched with a puncher to make a cake, and the cake was inoculated into pre-packaged and sterilized. In a good PDA liquid medium, shake culture on a shaker (120r/min, 27 °C) for 15 days. After the fungus is fully fermented, the fermentation broth and the hyphae ball are pulverized into a uniform mixture by a stirrer at 121 ° C. Sterilize for 20 minutes, cool and store in a refrigerator at 4 °C for use.

(2) The seedling rapid propagation medium was configured according to the following components: the fungal elicitor in step (1) 100 ml/L+N6 basal medium + Huabao 1 8 g/L + sucrose 30 g/L + agar 6 g/ L+ potato 50g/L+ banana 80g/L, sterilized at 121 ° C for 20 min for use.

Example 4

The fungus elicitor was added in an amount of 200 ml/L in the seedling rapid propagation medium, and the other procedures were the same as in Example 2.

Example 5

The fungal elicitors were used as control. After 90 days of seed sowing, the average bulb size of the seedlings of Examples 1, 2 and 3, the average plant height and the content of chlorophyll a, chlorophyll b and chlorophyll a+b were determined.

Table 1

It can be seen from Table 1 that the fungal elicitor prepared from the genus Pseudomonas sphaeroides JSNLBJ-001 was added to the white peony tissue culture medium, which can significantly promote the germination and growth of the white peony seed. After 90 days of seed sowing, Example 1 The white mites in 2, 3 have formed larger bulbs, higher plant height, and basically meet the requirements of transplanting tissue culture seedlings, while the control bulbs are only 0.31cm, the plant height is only 5.32cm, and the transplantables are not reached. Specifications. From the chlorophyll content, the medium supplemented with the fungal elicitor can significantly increase the chlorophyll content and promote its growth.

Claims

A method for preparing a fungal elicitor, which is characterized in that it is prepared from a genus of Bacillus licheniformis named Purpureocillium lilacinum JSNLBJ-001, and the preservation number is CGMCC NO :13690.
The method for preparing a fungal elicitor according to claim 1, comprising: inoculating the P. glabrata in a medium for fermentation culture, and pulverizing the fermentation broth and the hyphae after the fermentation culture is completed. Sterilize.
The method for preparing a fungal elicitor according to claim 2, wherein the medium is a PDA liquid medium.
The method for producing a fungal elicitor according to claim 2, wherein the fermentation culture has a temperature of 25 to 27 ° C and a time of 10 to 15 days.
A fungal elicitor prepared by the preparation method according to any one of claims 1 to 4.
A medium for rapid propagation of white peony seedlings comprising the fungal elicitor of claim 5.
The medium for rapid propagation of white peony seedlings according to claim 6, wherein the concentration of the fungal elicitor is 50 to 200 ml/L.
The medium for rapid propagation of white peony seedlings according to claim 7, wherein the composition comprises: N6 basal medium + said fungal elicitor + Huabao No. 1 4 to 10 g/L + 25 to 30 g/L Sucrose +5 ~ 6.5g / L agar + 50 ~ 80g / L potatoes + 80 ~ 100g / L banana.
A method for rapid propagation of white peony seedlings by using a fungal elicitor, comprising: cultivating a white peony seed in a medium containing the fungal elicitor according to claim 5, and cultivating the tissue culture seedling.
The method according to claim 9, wherein the culture condition is: temperature 25±5° C., illumination 1500-2500 Lx, illumination time 8-12 h/d, culture time 100 ~ 120 days.