CN111269841B

CN111269841B - New endophytic fungus TK815 and application thereof

Info

Publication number: CN111269841B
Application number: CN202010164610.0A
Authority: CN
Inventors: 谢玲; 龙艳艳; 张艳; 张雯龙; 农倩; 陈艳露; 廖仕同; 覃丽萍
Original assignee: INSTITUTE OF MICROBIOLOGY GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-10-27
Anticipated expiration: 2040-03-11
Also published as: CN111269841A

Abstract

The invention relates to the technical field of microorganisms, in particular to a new endophytic fungus TK815 and application thereof. The endophytic fungus TK815 is a new strain different from the prior art at present, is found in soil of a white hole crater of a boulder surrounding crater group in Happy county of Bai-color City, Guangxi, the TK815 is a new genus of Herpotichiella aceracea, a microorganism of the genus is named as Tiankengomelania guangxiense, and the strain TK815 is a model strain of the genus; the discovery of the new microorganism enriches available microorganism resources of endophytic fungi, and the strain has good growth promoting effect on dendrobium officinale.

Description

New endophytic fungus TK815 and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of microorganisms, in particular to a new endophytic fungus TK815 and application thereof.

[ background of the invention ]

Endophytic fungi (Endophytic fungi) are widely present in tissues and organs of healthy plants, and have extremely rich species diversity and functional diversity. As a group of microorganism resources which are not fully researched, the endophytic fungi resources are in a wide variety, including rare and special (or new classification units) strains, and the ecological functions of the endophytic fungi resources are discovered and revealed gradually. Researches show that the endophytic fungi can be symbiotic with the host in a reciprocal way, endow plants with excellent growth traits, and improve the disease and insect resistance of the host and the stress resistance in a stress environment, so that the use of chemical fertilizers and pesticides can be reduced. The fungi have the characteristics of wide ecological distribution, wide host range and separable pure culture, so that the fungi play more and more important roles in agricultural sustainable development and have wider application prospects.

The applicant has conducted intensive studies on endophytic fungi, and has continuously searched for new endophytic fungi, for example, endophytic fungi having application number 201410220964.7 "a DSE strain and its application in sugarcane production", which is isolated from mangrove plant in northern gulf of Guangxi, belonging to the genus Devrisia, and having an effect of promoting the growth of Dendrobium officinale; the endophytic fungi of 'endophytic fungi L-14 for preventing and treating banana wilt and application thereof' with application number 201610652927.2 is obtained by trapping and separating from soil by an applicant, belongs to Schizotech, and has the effect of preventing and treating banana wilt; an endophytic fungus with application number of 201410220965.1 [ DSE strain 24L-4 and application thereof in dendrobium officinale production ], which is obtained by separating from camellia leaf tissue by the applicant and belongs to Devrisia; at the same time, the industry contains many other endophytic fungi that applicants have discovered, such as application No.: 201410220965.1 "DSE Strain 24L-4 and its application in Dendrobium officinale production" all disclose the application of dark endophytic fungi in promoting the growth of Dendrobium officinale, however, the microorganism disclosed in 201710833508.3 is a species of Verticillium umbiliciformis (Zasmium sp.) and the microorganism disclosed in 201410220965.1 is a species of Devrisia sp.; because the endophytic fungi and a host have good reciprocal symbiotic relationship, the research on the endophytic fungi is not stopped at present, a plurality of new species of endophytic fungi which are not recognized and discovered by people exist in nature, and the research of the application is necessary for enriching the germplasm resources of the endophytic fungi.

[ summary of the invention ]

In view of the above, in order to enrich germplasm resources of endophytic fungi, screening, classifying and researching the endophytic fungi are necessary, and the applicant develops a new genus endophytic fungus TK815 which can be symbiotic with dendrobium officinale.

In order to achieve the aim, the invention screens out a new strain Tiankengomelania guangxienseTK815 with the preservation number of CGMCC NO:14346, which is preserved in China general microbiological culture Collection center on 29.06.2017, and the preservation addresses are as follows: institute of microbiology, national academy of sciences, Beijing, China.

Further, the strain is separated from soil by a plant trapping method.

Further, the soil is collected from an original forest of a white hole sky pit of a large stone surrounding sky pit group in Caulencian county of Guangxi Baishi county of a special habitat.

The invention also provides application of the new genus strain Tiankengomelania guangxiense TK815 in promoting angiosperm plant growth.

Further, the angiosperm is dendrobium officinale and/or sugarcane.

Further, the dendrobium officinale is a dendrobium officinale tissue culture seedling and/or a potted seedling, and the sugarcane is a sugarcane tissue culture seedling and/or a potted seedling.

The invention also provides a method for separating the new-genus strain, namely the Tiankengomelania guingxiense TK815, which comprises the steps of adopting Chinese cabbage as a bait plant to trap microorganisms in a soil sample, carrying out germination acceleration on Chinese cabbage seeds, mixing the Chinese cabbage seeds with the soil sample and a sterilized seedling culture substrate for culture, taking out the roots of the Chinese cabbage after one month, cleaning and disinfecting the roots of the Chinese cabbage, putting the Chinese cabbage on a separation culture medium flat plate, and transforming the Chinese cabbage onto a purification culture medium for purification after hypha grows out to obtain the strain.

Further, the separation medium is: 1/2 corn flour culture medium, which comprises the following components: 8.5g of corn flour agar, 15g of agar powder and distilled water to a constant volume of 1000 mL.

Further, the purification medium is: the CMMY culture medium comprises the following components: 10g of wort agar, 8.5g of corn flour agar, 2g of yeast extract, 7.5g of agar powder and distilled water to a constant volume of 1000 mL.

The invention also provides a method for promoting angiosperm TK815 to grow by applying the new strain Tiankengomelania guangxiense, which comprises the following steps: inoculating the strain TK815 into a tissue culture seedling and/or a plant of an angiosperm; the inoculation method is that the tissue culture seedling of the angiosperm plant is directly inoculated in a culture medium containing TK815 strain.

The invention has the following beneficial effects:

(1) the endophytic fungus TK815 is a new strain different from the prior art at present, and is found in soil of a large stone surrounding sky pit group white hole sky pit in Happy county of Bai color city of Guangxi, wherein the genetic relationship of the strain is very close to that of Herpotichiellaceae. The ITS +18S +28S sequence of representative species of each genus was selected under the family Herpotrichiellaceae to construct a phylogenetic tree together with the related sequence of the strain TK815, and the result showed that the strain TK815 formed an independent branch and did not come together with any known species; thus, TK815 was judged to be a new genus of the family Herpotichiella, which was named Tiankengomelania guangxiense and the strain TK815 was selected as a model strain of this genus. The discovery of the new microorganism enriches available microorganism resources of endophytic fungi, the strain has a good growth promoting effect on the dendrobium officinale, the strain is the endophytic fungi which is discovered for the first time in the region and has an obvious growth promoting effect on the dendrobium officinale, the blank of research in related fields at present is filled, and a certain guiding effect is played for the subsequent planting of the dendrobium officinale in the region. The TK815 strain provides resource guarantee for the development and utilization of soil endophytic fungi of original forests of the white hole sky pits of the Guangxi large stone surrounding sky pits in the production of the dendrobium officinale.

[ description of the drawings ]

FIG. 1 shows a phylogenetic tree constructed by strain TK815 based on ITS sequences

FIG. 2 is a morpholomorphic observation diagram of a strain of TK 815; wherein, Panel A shows the colony morphology of the strain; FIG. B-I shows conidia and conidiophores of the strains; a scale: a is 3 cm; B-I ═ 10 μm.

FIG. 3 is a control of growth after TK815 strain is inoculated to the dendrobium officinale seedling cultured on a plate; wherein, the left side represents the control plate seedling; the right side shows the plate seedling after TK815 inoculation;

FIG. 4 is a control of the growth of potted Dendrobium officinale seedlings inoculated with TK815 strain, where the left side shows the control potted seedlings; the right side shows the pot seedlings after TK815 inoculation.

[ detailed description ] embodiments

The invention provides a new strain Tiankengomelania guangxiense TK815 with the preservation number of CGMCC NO:14346, which is preserved in China general microbiological culture Collection center in 29.06.2017 at the preservation address: institute of microbiology, national academy of sciences, Beijing, China.

Example 1:

the novel genus strain, Tiankengomelania guangxiense TK815, is subjected to morphological identification and molecular biological identification, and specifically comprises the following steps:

(1) morphological identification:

as shown in part a of fig. 2, the colony morphology of the strain was observed as: colonies grew slowly on CMMY medium (wort medium) and had a colony diameter of 12-15mm after two weeks at 28 ℃. The colony is nearly circular, dark grey black, raised, with radial furrows.

As shown in part B-I of FIG. 2, the strain had smooth hyphae with septa, light brown green color, and a width of 1.6-3.4. mu.m. Conidiophores are upright, have separation, have no branch or 1 branch, are slightly thicker than hyphae, are light brown green and have spore forming marks of 4.6-87.8 multiplied by 1.7-3 mu m. Conidiophore stick-shaped, rough surface, blunt round end, truncated base, light brown green, 0-2 partitions, constriction at diaphragm, 5.1-14.9 × 1.1-2.4 μm.

(2) Molecular biological identification

DNA sequence BLAST was performed in GenBank database using sequenced ITS, 18S and 28S sequences, and revealed a close relationship to strain TK815 in the family Herpotrichiellaceae. The ITS +18S +28S sequence from each representative species of genus was selected under the family Herpotrichiellaceae to construct a phylogenetic tree together with the related sequence of the strain TK815, as shown in FIG. 1, the strain TK815 formed an independent branch and did not come together with any known species. From a morphological point of view, the strain TK815 has a short and rarely branched phimosis-producing peduncle, a slender and only 0-2 divided conidia, which combine to distinguish it from all known species in this family. As an independent branch is formed on the phylogenetic tree constructed by combining ITS +18S +28S three-section molecular fragments, the strain TK815 has a large genetic distance with closely related species, and the strain TK815 is identified as a new genus named as Tiankengomelania guangxiense by combining morphological differences.

Therefore, the strain TK815 can be proved to be a new genus strain of Herpotichiella family in terms of molecule and morphology, the classification status of the strain is Herpotichiella family, the strain is named as Tiankengomelaniaguengxiense, and the strain TK815 is selected to be a model strain of the genus strain.

Example 2:

the isolation method of the strain of example 1 is as follows:

(1) sampling

Sampling the soil (24 degrees in northern latitude 49 '2.95', 106 degrees in east longitude 27 '22.32', and 1175.7 meters in altitude) of the large stone surrounding sky pit group white hole sky pit in Bai-color city, Guangxi, and collecting the original forest plant rhizosphere soil of the large stone surrounding sky pit group by a five-point sampling method, wherein the sampling is carried out 5-15cm away from the soil surface. The soil sample is treated as soon as possible after being collected or is placed in a refrigerator at 4 ℃ for standby.

S1: preparing an isolation medium and a purification medium:

the preparation method of the isolation medium (1/2 corn flour medium) comprises the following steps: weighing 8.5g of corn flour agar and 15g of agar powder, and finally adding distilled water to a constant volume of 1000 mL and mixing uniformly;

the preparation method of the purified culture medium (CMMY culture medium) comprises the following steps: weighing 10g of wort agar, 8.5g of corn flour agar, 2g of yeast extract and 7.5g of agar powder, and finally adding distilled water to a constant volume of 1000 mL and uniformly mixing;

s2: chinese cabbage is used as a bait plant to trap endophytic fungi in soil samples. Sterilizing the surface of Chinese cabbage seeds (respectively sterilizing by using 75% alcohol and 1% sodium hypochlorite through oscillation for 30s), placing the Chinese cabbage seeds on a water agar culture medium for accelerating germination, mixing the collected soil sample and a sterilized seedling culture substrate in a ratio of 2:1, placing the mixture into a culture cup, and transplanting the Chinese cabbage with accelerated germination into the cup for culture. The culture can be carried out for about 50 days after 4 times of treatment. Collecting the roots of Chinese cabbage seedlings, cleaning, sterilizing the surfaces of the roots (shaking with 0.005% Tween 20 and sterile water for three times, 1min each time), placing the roots on 1/2 corn flour culture medium, culturing at 25 ℃, and timely transferring the roots to CMMY culture medium after the mycelia grow out from the root parts.

Example 3: research on growth promotion condition of strain TK815 to plants

The method comprises the following steps of (I) promoting the growth of dendrobium officinale:

(1) growth promotion test of TK815 on dendrobium officinale tissue culture seedlings:

activating strain TK815, inoculating to 3 oat culture media (OMA, g/L, oat flour 10.0, agar 14.0, MgSO₄·7H₂O 1.0，KH₂PO₄1.5，NaNO₃1.0), 3 levels, each named separately: TK815-1, TK815-2 and TK815-3, inoculating 3 bacterial blocks in each dish, and growing bacterial colonies for later use. Selecting sterile Dendrobium officinale seedlings with basically consistent plant height and thriving degree, transplanting the seedlings to colonies, transplanting 1 sterile Dendrobium officinale seedling to each colony, transplanting 3 sterile Dendrobium officinale seedlings to each colony, placing the culture dish into a tissue culture bottle, and culturing at 26 ℃ under the illumination intensity of 180 mu mol/m²·s²And culturing in a climatic chamber with illumination time of 16 h/d. The dry weight index was measured 45d after inoculation (test results are shown in table 1).

TABLE 1 influence of TK815 on the growth of tissue culture seedlings of Dendrobium officinale

Treatment of	Dry weight (mg)
		TK815-1	141.2
TK815-2	151.2
		TK815-3	251.6
CK	112.3

As can be seen from the above table, after 45d of inoculation, the dry weight of the dendrobium officinale cultured by 3 groups of TK815 plates is far greater than that of the CK group, and the increase of the average dry weight of the TK815 treated group compared with the control group is 61.47%.

As shown in fig. 3, the left side of fig. 3 shows the growth of the tissue culture seedling of dendrobium officinale in the CK group, and the right side of fig. 3 shows the growth of the tissue culture seedling of dendrobium officinale in the TK815 plate, which is obviously superior to the CK group.

(2) Growth promotion test of TK815 to dendrobium officinale pot culture seedlings:

s1: planting and managing tissue culture seedlings: the selected tissue culture seedling varieties are as follows: the number is 784 provided by the flower research institute of Guangxi agricultural academy of sciences,

s2: the bacterium application method comprises the following steps: and selecting seedlings with consistent growth, wherein 3 seedlings are planted in each cup, and the bacteria pouring is started one month after the dendrobium officinale seedlings are planted. 4 repeats were set up, each 12 cups (each named TK815-1, TK815-2, TK815-3, and TK815-4, respectively); the bacterium application method comprises the following steps: the preparation method of the bacterial liquid comprises inoculating mycelium block into PDB, shake culturing in shaker at 26 deg.C and 100rpm for 14d, filtering with sterilized gauze to collect mycelium pellet, washing with sterilized water for several times, and crushing to obtain mycelium pellet with concentration of 5 × 10⁵CFU/mL of bacterial liquid. Irrigating the root of each dendrobium officinale seedling with 30mL of bacterial liquid once every 15 days for 3 times. In addition, non-pouring bacterial liquid is used as control treatment;

measurement indexes are as follows: after 6 months of first fungus watering, measuring the plant height, stem diameter and dry weight of the dendrobium officinale seedlings, wherein the specific conditions are shown in table 2:

TABLE 2 influence of TK815 on the growth of Dendrobium officinale pot culture seedlings

Treatment of	Plant height (cm)	Diameter of the stem (cm)	Dry weight (mg)
				TK815-1	7.4	6.7	456.6
TK815-2	8.2	7.6	393.3
				TK815-3	6.6	6.9	430.4
TK815-4	5.7	7.0	345.6
				Control	4.4	5.4	228.2

As can be seen from the table above, after 6 months of inoculation, the dry weight, the plant height and the stem diameter of 4 groups of pot-planted dendrobium officinale of TK815 are far greater than those of the CK group, and after 45 days, the increase of the average plant height of the TK815 treated group compared with the control group is 58.52%; the increase in stalk diameter evaluation was 30.56%; the average increase in dry weight was 78.12%.

As shown in FIG. 4, the left column in FIG. 4 is the growth condition of the CK group Dendrobium officinale pot seedlings, the right column in FIG. 4 is the growth condition of the TK815 plate inoculated Dendrobium officinale pot seedlings, and the TK815 plate inoculated Dendrobium officinale pot seedlings are significantly superior to those of the non-inoculated control group in growth vigor.

(II) growth promotion test of TK815 on sugarcane potted seedlings:

s1: the sugarcane seedlings are new Taitang No. 22 provided by the research institute of sugarcane, academy of agricultural sciences in Guangxi.

S2: hardening the sugarcane tissue culture seedlings in sand to grow to 5-7 leaves, selecting the sugarcane seedlings with consistent growth vigor, and trimming the leaves for planting. Transplanting the sugarcane seedlings into plastic flowerpots with the width of 20cm and the height of 19cm, planting 3 sugarcane seedlings in each flowerpot, pouring root fixing water on peat and fine river sand serving as culture media in a ratio of 1:3(v/v), and covering with plastic films for moisturizing for 7 days. And (5) transplanting 10d of the colonized post-pouring bacterial liquid. 3 replicates were provided, 12 cups each (each replicate was named TK815-1, TK815-2 and TK815-3, respectively). Inoculating a mycelium block into a potato glucose liquid culture medium (PDB), carrying out shake culture in a shaker at the rotating speed of 100rpm at 25 ℃ for 14 days, filtering and collecting mycelium pellets by using sterile gauze, washing the mycelium pellets by using sterile water for a plurality of times, and crushing the mycelium pellets to prepare a bacterial liquid with the concentration of 5 multiplied by 105 cfu/ml. Irrigating 50mL of bacterial liquid into the root of each sugarcane seedling, irrigating the root once every 15d, irrigating the root 3 times in total, and investigating the result 2 months after the root irrigation is finished. The treatment with the non-inoculated solution was used as a control, and a total of 12 seedlings were treated in 4 pots each. The test results are shown in table 3:

TABLE 3 Effect of TK815 on sugarcane potted seedlings

Treatment of	Dry weight (mg)
		TK815-1	6.5
TK815-2	7.2
		TK815-3	7.1
CK	5.1

It can be seen from the above table that the dry weight of the sugarcane cultured in 3 groups of the TK815 pot plants after 2 months of inoculation is much greater than that of the CK group, and the increase of the average dry weight of the TK815 treated group compared with the control group is 35.95%.

In summary, the strain Tiankengomelania guangxiense TK815 belongs to a new strain, can play a remarkable growth promoting role in plants, and is a crop growth promoting microorganism with good performance.

The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.

Claims

1. Bacterial strainsTiankengomelania guangxienseThe application of TK815 in promoting the growth of angiosperms is characterized in that the angiosperms are dendrobium officinale;

said strainTiankengomelania guangxienseTK815, the preservation number is CGMCC NO: 14346.

2. The strain of claim 1Tiankengomelania guangxienseThe application of TK815 in promoting angiosperm plant growth is characterized in that the dendrobium officinale is a dendrobium officinale tissue culture seedling and/or a pot culture seedling.

3. The strain of claim 1Tiankengomelania guangxienseThe TK815 method for promoting the growth of angiosperm is characterized in that the angiosperm is dendrobium officinale,the method comprises the following steps: inoculating the strain Tiankengomelania guangxienseTK815 into a tissue culture seedling of dendrobium officinale, wherein the inoculation method is to directly inoculate the tissue culture seedling of the dendrobium officinale into a culture medium containing the TK815 strain.

4. The strain of claim 1Tiankengomelania guangxienseThe TK815 is a method for promoting the growth of angiosperm plants, and is characterized in that the angiosperm plants are dendrobium officinale, and the method is to inoculate a strain Tiankengomelania guangxienseTK815 into dendrobium officinale plants.