CN115537346A

CN115537346A - Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof

Info

Publication number: CN115537346A
Application number: CN202211471187.4A
Authority: CN
Inventors: 秦姣; 许格格; 张石宝; 张伟; 杨颖婕
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2022-11-23
Filing date: 2022-11-23
Publication date: 2022-12-30
Anticipated expiration: 2042-11-23
Also published as: CN115537346B

Abstract

The invention relates to a fungus for promoting growth and differentiation of sansevieria trifasciata and application thereof, and discloses a fungus for promoting differentiation of original bulbs of sansevieria trifasciata into seedlings, wherein the fungus is classified as the fungus QS494 (Tulasnella sp.) and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.25305. The strain can remarkably promote the protocorm of the Tibet sansevieria to grow and differentiate quickly to form seedlings, has very important practical significance for obtaining and breeding the symbiotic seedlings of the Tibet sansevieria, has the characteristics of low cost, quick seedling formation and the like compared with aseptic seedling culture, has application value for replacing the aseptic seedling culture to obtain the symbiotic seedlings, and provides reliable technical guarantee for solving the bottleneck problem of the source of the seedlings in the Tibet sansevieria cultivation industry.

Description

Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof

Technical Field

The invention relates to the technical field of botany, in particular to a jelly fungi for promoting growth and differentiation of Tibet sansevieria and application thereof.

Background

Sansevieria trifasciata (Cymbidium tracynum) is a special flower resource in China and is mostly distributed in forest of south China. In the field, the species can be attached to the branches and trunks of various trees, and has the advantages of large plants, flowery colors, long flowering phase and high ornamental value. Tiger head orchid is an important parent of cymbidium hybrid lines such as huilan hybrida and the like, and seedlings are obtained mainly by introducing wild plants or seeds for sterile germination for seed selection.

The seeds of orchids have in themselves special biological properties, usually very fine, without endosperm. Under natural conditions, the germination of orchidaceous seeds into seedlings must rely on specific symbiotic fungi for nutrients, and the colonization of symbiotic fungi is also required in the root system of adult plants. The orchid can be propagated by natural tillering and aseptic seeding, the propagation coefficient of the orchid is low, and the propagation coefficient of the orchid depends on detailed exploration of required nutritional conditions. At present, cymbidium tibetanum can obtain seedlings through aseptic seeding, but the time and the technical cost of aseptic seedling culture are high, the steps are complicated, for example, different culture mediums are needed in the stages of seed germination (about 1 month), protocorm proliferation subculture (about 4 months) and rooting and seedling strengthening (1 month), and the seedlings need to be exercised for two weeks after being taken out of bottles and grow slowly and are susceptible to diseases. The orchid seedlings, namely the symbiotic seedlings or mycorrhizal seedlings, obtained by symbiotic culture with fungi only need relatively barren culture conditions, grow and develop quickly, and have stronger adaptability after being taken out of bottles. Because the symbiotic relationship between the orchid seeds and the fungi has specificity, the determination of effective fungi which can form the symbiotic relationship with the Tiger orchid and promote the growth and differentiation is a key link for cultivating mycorrhizal seedlings. At present, related patents related to application of symbiotic fungi in promoting growth are provided aiming at medicinal orchidaceae plants such as dendrobium, bletilla and pleione, but reports of application and methods of the fungi in promoting differentiation of cymbidium seedlings are still lacked.

Disclosure of Invention

In order to solve or partially solve the problems in the related art, the invention provides the colletotrichum gloeosporioides for promoting the growth and differentiation of the sansevieria trifasciata and the application thereof. After purifying and culturing the glued membrane bacterial strain QS494 on a PDA plate culture medium, inoculating the glue membrane bacterial strain QS494 and the sansevieria trifasciata protocorm to the surface of a symbiotic culture medium at the same time, and after about two weeks, enabling the protocorm to form symbiotic relation with fungi. After five weeks, the growth and differentiation of the inoculated and non-inoculated protocorms showed large differences. Three months later, the Tibet sansevieria seedlings inoculated with the cellophane QS494 grow strongly, and 90% of individuals differentiate into roots. Compared with a non-inoculated fungus group and a Piriformospora indica inoculation group, the sansevieria trifasciata inoculated with the mucilaginosus QS494 has remarkable advantages in seedling height and individual weight, and the fact that the mucilaginosus QS494 has a strong promoting effect on growth and differentiation of the sansevieria trifasciata is proved.

The first aspect of the present invention provides a colletotrichum QS494 (tulasella sp.), with a collection number of: CGMCC No.25305.

The second aspect of the invention provides application of the hymenophora QS494 to breeding of Tibet sansevieria.

In a third aspect of the present invention, there is provided a method for promoting differentiation of native bulbs of sansevieria trifasciata into young plants, comprising the steps of:

the preservation numbers are: culturing the Mycoleptodonoides Chevr QS494 of CGMCC No.25305 in a culture dish, cutting into bacterial pieces, sowing the bacterial pieces and sterile protocorm of sansevieria trifasciata on a symbiotic culture medium, and culturing at 25 ℃ in an artificial climate chamber to obtain symbiotic seedlings.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

The invention has the beneficial technical effects that:

(1) The invention selects two orchid mycorrhizal fungi strains (Piper indica and Mucor pulchrum strain QS 494) for carrying out symbiotic culture with the original bulb of the Tibet sansevieria, wherein the Mucor pulchrum strain QS494 can promote the original bulb of the Tibet sansevieria to be rapidly differentiated into high-quality symbiotic seedlings with the leaf length of about 2 cm and the root length of about 1 cm in three months, and compared with aseptic seedling culture, the time is shortened by two months.

(2) The Thamnolia vermicularis (Tiger) Ascens seedling culture is carried out on a barren culture medium by adopting Mucor pulchrum QS494, and the protocorm is only transferred once after being formed. Compared with the prior art, the sterile seedling culture needs to be transferred to a proliferation subculture medium firstly, and then transferred to a rooting and seedling strengthening culture medium after four months, and the two culture media are complicated in nutrient components and need to be added with hormones. Therefore, the strain is used for symbiotic seedling raising, the labor and resource cost can be greatly reduced, the strain is suitable for popularization and application, and the strain has an application value of replacing aseptic seedling breeding.

Description of biological preservation

Tulasnella sp.qs494, classification nomenclature: tulasnella sp (Mycoleptomycetales, agaricaceae, basidiomycota), collected at the general microbiological center of China Committee for culture Collection of microorganisms, no. 1 Siro-Lu, north-West district, chaoyang, beijing, on day 07, 15 of 2022, with the collection number of CGMCC No:25305.

drawings

FIG. 1 shows morphological characteristics of hypha and moniliform cells of Mycoplasma gondii QS494 (CGMCC 25305) in the present invention. The bright spots in the hyphal cells are nuclei and the white arrows indicate cell septa. And dyeing by using Hoechst 33342 dye liquor, and observing under a fluorescence microscope.

FIG. 2 shows the Thomas tibetana protocorm 15 days after inoculation into OMA medium in the present application. The left, middle and right are respectively an inoculation-free control group, an inoculation hymenophora QS494 control group and an inoculation Piriformospora indica control group. Arrows indicate fungal mycelial mass.

FIG. 3 shows comparison of growth of Thomas tibetana protocorm in the present application after 35 days of inoculation into OMA medium. The left, middle and right are respectively an inoculation-free control group, a colletotrichum QS494 inoculation group and an Indian pyricularia inoculation group.

FIG. 4 comparison of growth of sansevieria trifasciata protocorm in the present application after 90 days of inoculation into OMA medium. The upper and lower panels on the left are non-inoculated control groups, the upper and lower panels in the middle are inoculated groups of collectins QS494, and the upper and lower panels on the right are inoculated groups of Piriformospora indica.

FIG. 5 is a comparison of the plant height (left panel) and individual weight (right panel) of a Tibet sansevieria seedling of the present application, measured 90 days after the protocorm was transferred to OMA medium. Asymbiotic, symbio-QS494 and Symbio-Si are respectively used as a non-inoculated control group, a colletotrichum QS494 inoculated group and an Indian pyricularia inoculation group. Plant height was measured using Image J software, and root length was not taken into account. Data are mean + standard error (statistical population > 40), and the letters (A-C) above the histogram are different, indicating significant difference between groups (one-way analysis of variance, LSD test, P < 0.05). Analysis of variance was done in SPSS16.0 and histograms were made by SigmaPlot 14.0 software.

Detailed Description

Alternative embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While the drawings show alternative embodiments of the present application, it should be understood that the present application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

The invention discloses a jelly fungi for promoting growth and differentiation of sansevieria trifasciata and application thereof, which are described in detail in the following with reference to the accompanying drawings:

mucor (tunasella sp.) QS494, its deposit number is: CGMCC No.25305.

The colony morphology is milky white, the colony grows radially, hyphae are binuclear, and moniliform cells are easy to generate (figure 1).

The strain acquisition pathway is as follows: the orchid mycorrhiza segments are taken and transversely cut into slices under a dissecting mirror, and the slices are placed under a microscope to observe the colonization condition of a fungal mycelial mass (peloton). Placing the mycorrhiza segments rich in mycelial clusters in a sterilized triangular flask containing 2% NaClO, soaking for 4 min, taking out with tweezers, and soaking and washing with sterile water for 3 times. On a super clean bench, a mycorrhiza segment is obliquely cut by a sterile scalpel, the section is attached to a PDA culture plate, and the culture is carried out in an inverted mode at 25 degrees. And (4) cutting hyphae to a new transfer culture medium after the hyphae grow on the edge of the mycorrhiza fragment, and numbering to obtain pure colonies. Scraping hyphae on the surface of a plate culture medium, extracting DNA by using a CTAB method, and performing PCR amplification and DNA sequencing identification. And sterilizing the prepared PDA culture medium, subpackaging into sterile 18 x 180 mm glass test tubes with silica gel plugs, wherein the subpackaging amount of each tube is 1/3 of that of the test tube, and placing on a slope for cooling for later use. Inoculating the mycelia of the purified strain cut by a sterile scalpel on a slant of a test tube on a super clean bench, and noting the serial number and date of the strain. Placing the inoculated test tube in an incubator for culture at 25 ℃, transferring the test tube to 4 ℃ for preservation when hyphae are about to grow over the slope, making copies and sending the copies to the bacterial preservation center of the institute of microbiology of Chinese academy of sciences for long-term preservation.

The method for promoting the differentiation of protocorms of sansevieria tibetana to form seedlings is characterized by comprising the following steps:

and (3) performing purification culture on the cellophane strain QS494 in a PDA (personal digital assistant) plate culture medium, then simultaneously inoculating the cellophane strain QS494 and protocorms of the Tibet sansevieria to the surface of a symbiotic culture medium, and forming a symbiotic relationship between the protocorms and fungi after about two weeks. Five weeks later, the growth and differentiation of the inoculated and non-inoculated protocorms showed large differences. Three months later, the Tiger head orchid seedlings inoculated with the Mucor Tinctorii QS494 grow strongly, and 90% of individuals differentiate into roots. Compared with a non-inoculated fungus group and a Piriformospora indica inoculation group, the sansevieria trifasciata inoculated with the mucilaginosus QS494 has remarkable advantages in seedling height and individual weight, and the fact that the mucilaginosus QS494 has a strong promoting effect on growth and differentiation of the sansevieria trifasciata is proved.

For the sake of clarity, the following examples are given in detail.

Example 1 effectiveness experiment of Mucuna collettii strain QS494 for promoting growth and differentiation of protocorm of Tibet sansevieria:

1. strain activation: on a clean bench, a small piece of the strain refrigerated in the early stage is cut to the center of a newly prepared culture plate, the plate is sealed and numbered, and the plate is inversely cultured for one week in the dark condition to recover the activity of the strain. The strain activation adopts a Potato glucose medium (PDA) culture medium, and the preparation method is as follows: decocting 200 g of peeled potato to soften, collecting filtrate, adding 20 g of glucose and 15 g of agar powder, boiling to constant volume to 1L, subpackaging to a triangular flask, sterilizing at 121 ℃ for 15 min under high pressure, subpackaging to a culture dish, and cooling for later use.

2. Obtaining Tiger head orchid protocorm: placing Tiger-head-orchid seeds in MS culture medium (Murashige & Skoog Medium，sigma-aldrich) And (3) performing sterile sowing to obtain protocorms (the specific method can refer to the 2009 plant physiological communication 45 of royal jelly and the like.

3. The symbiotic culture experiment of fungi and Tibet sansevieria protocorm adopts Oat Agar medium (OMA, 4 g/L of Oat, 8 g/L of Agar, boiling to constant volume, sterilizing, and packaging for use). In the clean bench, tiger Tibet orchid aseptic protocorm is spread by tweezers and inoculated on OMA culture medium. The first step activated fungal excised discs were inoculated into the center of an OMA plate. The experiment was performed with a sterile control group, QS494 inoculation group and Piriformospora indica inoculation group. The sterile control group was treated in the same manner as the fungal inoculation group except that the center of the OMA culture plate was inoculated with a sterile culture medium. Piriformis indica (L.) (Serendipita indica) Belonging to order Serrendipitaceae of Cereus of Basidiomycota, and showing stronger growth promoting effect on part of orchids (such as hybrid of orchid genus and Dendrobium officinale, etc.) (see the detail in southeast gardening No. 2020, lu and billows, etc. 20; schlemm 29, san Fenlai et al 2021 tropical and subtropical plant school 29). Meanwhile, an Indian pyricularia piriformis inoculation group is arranged, so that the promotion effect of the cellophane strain QS494 on the differentiation and seedling formation of the protocorm of Tibet sansevieria can be intuitively reflected. The Piriformospora indica strain used in the experiment is purchased from the common microorganism center of China Committee for culture Collection of microorganisms (the preservation number is CGMCC No. 3.17686). All three treatment groups are placed in a culture room with the light intensity of 1000 Lx for 12 h/dark 12 h, and cultured at the constant temperature of 25 ℃.

4. The culture dish is regularly observed, and after two weeks, fungal hyphae can be seen to be distributed on the surface of the culture dish, and protocorms are kept in a healthy state. The protocorm is taken for anatomical observation, and symbiotic fungal mycelial masses (figure 2) are found to be colonized in the basal cortex cells of the protocorm of the two fungal inoculation groups, which shows that the two fungi can form a symbiotic relationship with Tibet sansevieria.

5. After five weeks, the sterile protocorms and the inoculum protocorms showed visual differences in growth and differentiation, which were specifically shown as: there was no significant change in the sterile protocorm from the initial stage of inoculation, but 1-2 more young leaves were differentiated from the inoculated protocorm (FIG. 3).

6. Three months after inoculation, the sterile protocorm and the inoculated protocorm showed large differences in growth and differentiation. The concrete expression is as follows: the non-inoculated control group differentiated into leaves, and of more than 40 protocorms, about 33% of individuals differentiated into roots, but the plants were smaller; the Mucor QS494 inoculation group and the Piriformospora indica inoculation group have better seedling leaf growth vigor, but 90 percent of individuals in the former treatment group differentiate roots, and only 7.5 percent of individuals in the latter treatment group differentiate shorter roots. Through measurement and statistics of seedling height and individual weight, the Tibetan sansevieria trifasciata protocorm inoculated with the tunica albuginea QS494 is found to have significant difference in growth and root differentiation from a non-inoculated control group (figure 5).

Having described embodiments of the present application, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. Mycoplasma donovani QS494 (Tulasnella sp.), characterized in that the preservation number is: CGMCC No.25305.

2. The application of the hymenomycetes QS494 in Tibet sansevieria seedling breeding according to claim 1.

3. The method for promoting the differentiation of the protocorm of the Tibet sansevieria to form the seedling is characterized by comprising the following steps:

the preservation numbers are: muscolecodon QS494 of CGMCC No.25305 is activated and cultured in a culture dish, then a strain sheet is cut, and the strain sheet and sterile protocorm of sansevieria tibetana are simultaneously sown on a symbiotic culture medium, and symbiotic seedlings are obtained under the culture condition of 25 ℃ in an artificial climate chamber.