CN110438017B

CN110438017B - Cladosporium cladosporioides strain LJ1 and application thereof

Info

Publication number: CN110438017B
Application number: CN201910884589.9A
Authority: CN
Inventors: 农倩; 谢玲; 陈艳露; 张艳; 廖仕同; 覃丽萍
Original assignee: INSTITUTE OF MICROBIOLOGY GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: INSTITUTE OF MICROBIOLOGY GUANGXI ZHUANG AUTONOMOUS ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2019-09-19
Filing date: 2019-09-19
Publication date: 2020-04-10
Anticipated expiration: 2039-09-19
Also published as: CN110438017A

Abstract

The invention relates to the technical field of biology, in particular to a Cladosporium cladosporioides strain LJ1 and application thereof. The invention discloses a Cladophiaphora sp.LJ1 of Cladosporium with a preservation number of CGMCC NO.18140, which can effectively prevent and treat banana vascular wilt, wherein the prevention and treatment effect in a dish symbiotic experiment reaches 88.89% and the prevention and treatment effect in a pot experiment reaches 53.67% in a resistance experiment of the banana vascular wilt; meanwhile, the strain has broad-spectrum growth promoting effect, and can effectively promote the growth of tomato seedlings (solanaceae plants) and dendrobium officinale seedlings (cyanobacteria plants).

Description

Cladosporium cladosporioides strain LJ1 and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of biology, in particular to a Cladosporium cladosporioides strain LJ1 and application thereof.

[ background of the invention ]

In recent decades, the use of chemical fertilizers and pesticides has greatly promoted the increase and stable yield of grains, but the long-term unreasonable use of chemical fertilizers and pesticides seriously threatens the safety of environment and agricultural products. Therefore, the search for new agricultural methods and technologies with high efficiency and good environmental compatibility is an urgent need to solve the current environmental pollution and food safety crisis. And the growth promotion and disease and insect resistance functions of the microorganisms in the ecological system on crops are fully utilized and exerted, and the ecological and economic significance is provided for improving the production capacity of the crops, protecting and improving the agricultural ecological environment and promoting the sustainable development of agriculture. 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. The applicant has obtained good results in the field of research on endophytic fungi, such as the endophytic fungi with the application number of 201410220964.7 entitled "a DSE strain and its application in sugarcane production", which is isolated from mangrove plants in northern gulf of Guangxi, belongs to the genus Devrisiasp, and has the effect of promoting the growth of Dendrobium officinale; the endophytic fungus is an endophytic fungus L-14 for preventing and treating banana wilt and an application thereof under the invention name of 201610652927.2, is obtained by trapping and separating from soil by an applicant, belongs to Schizotech, and has the effect of preventing and treating the banana wilt; therefore, the endophytic fungi has wide development and utilization prospects in agricultural natural environment protection. In order to fully exploit the ecological functions of endophytic fungi, more excellent strains need to be further screened from abundant and diverse endophytic fungi germplasm resources, so that resource guarantee is provided for development and utilization of the strains.

[ summary of the invention ]

In view of the above-mentioned discovery of more endophytic fungi with excellent properties and the promotion of the development and utilization of endophytic fungi germplasm resources, the present invention aims to provide an aspergillus cladosporioides strain (Cladophora sp.) LJ1 and its application in promoting the growth of angiosperms and preventing and treating banana vascular wilt.

In order to achieve the aim, the invention screens out a Cladophora sp strain LJ1 with the preservation number of CGMCC NO. 18140. Belongs to the kingdom fungi, the genus Cladophiaphora, unknown species. The endophytic fungi is preserved in China general microbiological culture Collection center, and the address is as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, the area of the rising of the south of the morning has the preservation number of CGMCC No.18140 and the preservation date of 2019, 07-month-04-day.

The colony morphology of the strain on a PDA plate culture medium is shown in figure 1 and figure 2, the diameter of the colony is about 15mm after the strain is cultured for 10 days at 25 ℃, the colony is circular to nearly circular, brown, white with impurities, felty and does not produce conidia.

Further, the strain is separated from the sugarcane root soil.

The invention also comprises a method for separating Cladophora sp strain LJ1, which is characterized in that sterile tomato seedlings are used as trapping plants and planted in the soil surrounding the roots of sugarcane for trapping, and after trapping is finished, strain separation is carried out on root tissues of the tomato seedlings by adopting 1/2CM culture medium to obtain the strain LJ 1.

Further, the 1/2CM medium comprises the following components: 8.5g of corn flour agar, 15g of agar powder and 1000mL of distilled water.

The invention also comprises the application of the Cladophora sp strain LJ1 in preventing and treating blight.

Further, the wilt disease is banana wilt disease.

The invention also includes the use of the above-mentioned strain LJ1 of the genus Cladophora sp for promoting the growth of angiosperms.

Further, the angiosperm is selected from tomato and/or dendrobium officinale.

The invention also comprises a method for preventing and treating banana wilt by applying the Cladophora sp strain LJ1, wherein the method comprises the following steps: inoculating the strain LJ1 into banana tissue culture seedlings and/or plants; the inoculation method comprises the step of directly inoculating the banana tissue culture seedlings in a culture medium for culturing the LJ1 strain and/or irrigating the activated LJ1 bacterial liquid to the roots of the banana tissue culture seedlings and/or plants.

The invention also comprises a method for promoting the growth of angiosperms by applying the Cladophora sp strain LJ1, which comprises the following steps: inoculating the strain LJ1 into a tissue culture seedling and/or a plant of an angiosperm; the inoculation method is to directly inoculate the tissue culture seedling of the angiosperm in a culture medium for culturing the LJ1 strain.

The invention has the following beneficial effects:

the strain Cladosporium sp (Cladophiaphora sp.) LJ1 can effectively prevent and treat banana wilt, the prevention and treatment effect in a dish symbiotic experiment in a resistance experiment of banana wilt is 88.89%, and the prevention and treatment effect in a pot experiment is 53.67%; meanwhile, the strain has a broad-spectrum growth promoting effect, and the growth of tomato seedlings (solanaceae plants) and dendrobium officinale seedlings (cyanobacteria plants) can be effectively promoted after the LJ1 is inoculated to the tomato seedlings and the dendrobium officinale seedlings.

[ description of the drawings ]

FIG. 1 is a back view of the colony morphology of the LJ1 strain of the present invention grown on PDA medium;

FIG. 2 is a front view of the colony morphology of the LJ1 strain of the present invention grown on PDA medium;

FIG. 3 is a graph of the ITS phylogenetic tree of LJ1 according to the present invention;

FIG. 4 is a graph showing the growth promoting effect of the LJ1 strain of the present invention on tomato seedlings; on the left in the figure are CK groups that were not inoculated with LJ 1; treatment groups inoculated with LJ1 are on the right;

FIG. 5 is a graph of the growth promoting effect of the LJ1 strain on Dendrobium officinale seedlings; on the left in the figure are CK groups that were not inoculated with LJ 1; treatment groups inoculated with LJ1 are on the right;

FIG. 6 shows the colonization of tomato roots by LJ1 of the present invention.

[ detailed description ] embodiments

The invention is further illustrated below with reference to the figures and examples and tests.

Example 1:

the invention provides a Cladophora sp strain LJ1 with the preservation number of CGMCCNO.18140. Belongs to the kingdom fungi, the genus Cladophiaphora, unknown species. The endophytic fungi is preserved in China general microbiological culture Collection center, and the address is as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, the area of the rising of the south of the morning has the preservation number of CGMCC No.18140 and the preservation date of 2019, 07-month-04-day.

The above-mentioned Cladosporium moulds were collected and isolated from sugarcane root soil (N24 DEG 01 '25.86', E109 DEG 24 '59.97', altitude 95.5 m) in the sugar cane field of Chuanshan town of Yangjiang county, Liuzhou, Guangxi.

The separation method adopts a trapping method, specifically adopts sterile tomato seedlings as trapping plants, plants the trapping plants in the soil around the roots of the sugarcane, and separates strains from the root tissues of the tomato seedlings by adopting 1/2CM culture medium after trapping to obtain the strain LJ 1.

The crop growth promoting endophytic fungi obtained by the separation is detected as follows:

1. morphological Classification of strains

The colony morphology of the strain on PDA plate culture medium is shown in FIG. 1 and FIG. 2, and the colony diameter is about 15mm after culturing at 25 deg.C for 10 days, and the strain is round to nearly round, brown, white, felt, and does not produce spore

2. Molecular biological identification

PCR was performed directly using fungal hyphae using a TIAngel one-tube convenient MasterMix kit. The ITS1-5.8S-ITS4-28S sequence (ITS + LSU) of rDNA is amplified by using universal primers (such as the sequence table SEQ ID NO:1-4) ITS1(5- 'TCCGTAGGTGAACCTGCGG-3') and LR5(5- 'TCCTGAGGGAAACTTCG-3'); NS1(5- 'GTAGTCATATGCTTGTCTC-3') and NS4(5- 'CTTCCGTCAATTCCTTTAAG-3') amplify the 18S rRNA (SSU) sequence of rDNA.

The PCR reaction system is as follows: TIAngel one tube convenient MasterMix 9 muL, 20 mumol/L positive and negative primers each 1 muL, template DNA 1 muL, ultrapure water constant volume to 25 muL.

The PCR reaction program is: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 53 ℃ for 1min, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃ for 10 min. Sequencing of PCR amplification products was performed by Shanghai Bioengineering technology, Inc.

Correcting and shearing the sequencing result to finally obtain an ITS sequence of 597bp, an LSU sequence of 907bp and an SSU sequence of 1068bp, comparing the obtained sequences on an NCBI website by using a BLASTN program, and displaying that the similarity between the ITS sequence and Cladophiaphora sp is 98.49% by using the comparison result; the similarity of the LSU sequence to Cladophiaphora sp. was 99.78%; the similarity of the SSU sequence to Cladophiaphora sp. was 99.78%;

step (5), clustering analysis: the ITS sequence was used to construct a phylogenetic tree (as shown in FIG. 3), and it was found that the strain was integrated with the Cladophora chaetospira strain (CBS 114747, CBS491.70, CBS514.63) at a support rate of 70%, but since the strain was not sporulating, species could not be identified in view of the identification information currently held, and thus, it was identified as an unknown species of the Cladophora genus.

Example 2:

the prevention and treatment effects on banana vascular wilt are as follows:

1. plate control effect:

(1) co-culture of LJ1 and banana tissue culture seedlings:

activating strain LJ1, inoculating to OMA culture medium (MgSO4 & 7H2O 1.0.0 g, KH2PO41.5g, NaNO31.0g, oat powder 10g, agar powder 11g, and distilled water 1000 mL), and culturing for 10 d; then selecting aseptic banana tissue culture seedlings with consistent growth vigor and transplanting the aseptic banana tissue culture seedlings onto bacterial colonies, transplanting 1 tissue culture seedling to each bacterial colony, transplanting 3 tissue culture seedlings to each dish, and naming the bacterial colonies as a treatment group; meanwhile, the banana tissue culture seedlings are transplanted to culture plates (3 per plate) without being inoculated with LJ1, and the culture plates are named as CK groups; and (3) putting the treatment group and the CK group into an incubator to perform co-culture under the same conditions: at 26 ℃ and the illumination intensity is 180 mu mol/m²·s²The illumination time is 16 h/d; the incubation time was 20 days.

(2) Preparation of a pathogenic culture medium:

activating fusarium (pathogenic bacteria), inoculating to a water agar culture medium, inoculating 3 bacterial blocks to each dish, and culturing at 28 ℃ for 4 days to obtain a disease treatment culture medium for later use.

(3) Plate control experiment:

transplanting the LJ 1-banana tissue culture seedlings (treatment group) obtained in the step (1) and the banana tissue culture seedlings (CK group) without the LJ1 into the pathogenic culture medium in the step (2), and then putting the seedlings into an incubator at 28 ℃ and with the light intensity of 180 mu mol/m²·s²And culturing for 15d under the condition of 80% humidity. Controls were directly plated onto water agar plates with fusarium. Observing and recording the disease grade of each treatment, calculating disease index and preventingHas therapeutic effect.

The disease grading criteria for plate seedlings are as follows:

grade 0-no visible symptoms in appearance;

grade 1-banana plants have no obvious resistance to growth, and the whole plant verticillium area is not more than 35%;

grade 2-banana plants are weak and small, and the verticillium wilt area of the whole plants reaches 35-80 percent;

the whole plant verticillium wilt area of 3 grade banana plant is up to more than 80%, even the whole plant withers.

2. The potted plant control effect is as follows:

(1) preparation of LJ1 bacterial liquid

Inoculating activated LJ1 into PDB liquid culture medium, shake culturing at 25 deg.C and 120rpm for 14d, filtering with sterilized gauze to collect mycelium, washing with sterilized water for several times, and pulverizing to 5 × 10⁵cfu·mL^-1The bacterial liquid of (4). .

(2) Preparation of pathogen suspensions

Fusarium is activated and then prepared into the product with the concentration of 1 × 106cfu & mL^-1The spore liquid of (1).

(3) Potted plant control effect experiment:

the experiment is carried out in a microbiological research institute network of Guangxi agricultural academy of sciences; uniformly planting banana seedlings (3-4 leaves) with consistent growth vigor in a nutrition cup, and planting the banana seedlings by adopting disease-free natural soil; dividing the banana seedlings into two groups, wherein one group is a treatment group, and the other group is a control group (CK group); then, 50mL of LJ1 bacterial liquid obtained in the step (1) is irrigated to the roots of the banana seedlings in the treatment group; synchronously pouring 50mL of distilled water into a control group (CK); then irrigating once every 15 days according to the same treatment method, and irrigating roots three times. After the last root irrigation is finished for 10 days, simultaneously carrying out root injury inoculation on the banana seedlings in the treatment group and the CK group to obtain fusarium spore suspension; culturing in artificial climate incubator, observing disease occurrence condition of banana seedling plant under the same culture condition, dissecting banana plant corm 25d after inoculation, observing and recording banana seedling disease grade, and calculating control effect, wherein each treatment is repeated for 3 times, and each repetition is 10 banana seedlings.

The disease grading standard for potted seedlings is as follows:

grade 0-healthy bulb without discoloration;

level 1: the color change area of the bulb accounts for less than 20% of the area of the bulb;

grade 2-the area of change of color of the bulb accounts for 20-40% of the area of the bulb;

grade 3-the area of the bulb that changes color accounts for 40% -60% of the area of the bulb;

grade 4-the area of change of color of the bulb accounts for 60-80% of the area of the bulb;

grade 5-the discoloration area of the bulb accounts for more than 80% of the area of the bulb, even the entire bulb is discolored and necrotized.

The prevention effect calculation formulas of the plate prevention and control effect and the potted plant prevention and control effect are the same, and the calculation formulas are as follows:

through the calculation of the formula, the control effect of the experimental strain LJ1 on banana vascular wilt is shown in Table 1:

TABLE 1 prevention and treatment effect of the strain LJ1 on banana vascular wilt

Treatment of	Mean index of disease in the plate	Average plate control effect	Average potted disease index	Average pot control effect
					LJ1	0.11	88.89％	0.17	53.67％
CK	1.00	—	0.37	—

As can be seen from the table 1, the resistance of the bananas to banana wilt can be improved by inoculating the LJ1 strain in the bananas, the control effect in a dish symbiotic experiment reaches 88.89%, and the control effect in a pot experiment reaches 53.67%.

Example 3:

growth promoting effect on tomatoes:

the method comprises the following steps:

(1) culturing an aspergillus oryzae strain LJ1 by using an OMA culture medium to obtain an LJ1 colony;

(2) carrying out surface disinfection and pregermination on plump tomato seeds for two days, then transplanting the plump tomato seeds to LJ1 bacterial colonies cultured in the step (1), and respectively transplanting 1 aseptic seedling on each bacterial colony, wherein each dish comprises 3 seedlings; the group of dishes inoculated with LJ1 was designated as the treatment group; meanwhile, synchronously transplanting aseptic seedlings to a blank OMA culture medium which is not inoculated with the strain in the step (1), and naming 3 seedlings in each dish as a CK group;

(3) putting the treated group in the step (2) and the CK group into an incubator together for co-culture, wherein the culture temperature is 26 ℃, and the illumination intensity is 180 mu mol/m²·s²The illumination time is 16 h/d.

(4) Repeating each treatment of the above step (2) 4 times; and observing the growth condition of the tomato seedlings after 14 days, cleaning the roots of the tomato seedlings, putting the cleaned tomato seedlings into an oven at 50 ℃, baking the cleaned tomato seedlings for 3 days, and weighing the dry weight.

Experiments show that the growth conditions of tomato seedlings after 14 days are shown in figure 4, the left side in the figure 4 is a CK group which is not inoculated with LJ1, the right side in the figure 4 is a treatment group which is inoculated with LJ1, the obvious growth condition of the tomato seedlings in the right side is obviously better than that in the left side, and the fact that the LJ1 can effectively promote the growth of tomatoes is demonstrated.

The dry weight results are shown in table 2:

TABLE 2 Effect of the growth promoting strains of the present application on tomato plantlet growth

Treatment of	Dry weight (mg)
		Treatment group	9.5
CK	8.3

As can be seen from Table 2, the dry weight of the treated group was significantly higher than that of the CK group, indicating that the strain LJ1 of the present application was effective in increasing the dry weight of tomatoes and promoting the growth of tomatoes.

FIG. 6 is a graph of the colonization of LJ1 on tomato roots, and it can be seen that there is a large number of hyphae distributed and colonized in the tomato root cell tissues, which indicates that LJ1 can be well colonized in the tomato body and can establish a good reciprocal symbiotic relationship.

Example 4:

the growth promoting effect on the dendrobium officinale:

(1) activating test strain, inoculating to OMA (oat flour 10g/L, agar 18g/L, MgSO)₄·7H₂O 1g/L，KH₂PO₄1.5g/L，NaNO₃1g/L), inoculating 3 bacterium blocks into each dish, and culturing for 10d until the bacterial colony grows up for later use;

(2) selecting sterile dendrobium officinale tissue culture seedlings with consistent growth vigor, transplanting the sterile dendrobium officinale tissue culture seedlings to bacterial colonies, transplanting 3 tissue culture seedlings on 3 bacterial colonies of each dish respectively, and naming the culture dish inoculated with the LJ1 strain as a treatment group; meanwhile, synchronously inoculating the sterile seedlings to a blank oat culture medium without inoculating LJ1 (the components of the culture medium are completely consistent with those in the step (1)), and naming the blank oat culture medium as CK group;

(3) putting the treated group and the CK group in the step (2) into an incubator for co-culture under the culture condition of 26 ℃ and the illumination intensity of 180 mu mol/m²·s²The illumination time is 16 h/d.

(4) The above step (2) is carried out 4 times of repeated treatments, after culturing for 60d, the growth indexes such as plant height are measured, the root culture medium is washed and then baked in an oven at 50 ℃ for more than 3d, and the dry weight is measured.

Experiments show that after 60 days, the growth condition of the dendrobium officinale seedlings is shown in fig. 5, the left side in fig. 5 is a CK group which is not inoculated with LJ1, the right side in fig. 5 is a treatment group which is inoculated with LJ1, the obvious growth vigor of the dendrobium officinale seedlings in the right picture is obviously superior to that in the left picture, and the fact that the LJ1 can effectively promote the growth of the dendrobium officinale is demonstrated.

In the embodiment, the plant height, stem diameter, root length, tillering, fresh weight and dry weight of the dendrobium officinale are measured or calculated; and analyzed using Microsoft excel 2003 and DPS version 7.05 data statistical analysis software, using Student's T test for comparison. The experimental results obtained are shown in table 3:

TABLE 3 influence of the Strain LJ1 on the growth of tissue culture seedlings of Dendrobium officinale

Treatment of

Plant height (cm)

Diameter of the stem (mm)

Root length (cm)

Tillering (one)

Fresh weight (g)

Dry weight (g)

Treatment group

4.11±0.59

2.25±0.29

6.98±.21*

2.11±1.13

0.48±0.24

0.14±0.05

CK

3.75±1.01

1.84±0.61

4.54±1.76

1.94±0.49

0.39±0.10

0.09±0.03

Description of the drawings: data in the same column indicates that the difference was up to a 5% significant level.

As can be seen from Table 3, the plant height, stem diameter, root length, tillering, fresh weight and dry weight of the treated group are all superior to those of the CK group, which indicates that the Cladosporium LJ1 of the application can effectively promote the growth of Dendrobium officinale.

To sum up, the Cladosporium cladosporium LJ1 belongs to a new strain, can play a role in preventing and treating banana wilt, can effectively promote the growth of angiosperms such as tomatoes, dendrobium officinale and the like, is a plant beneficial endophytic fungus with good performance, and has the functions of disease resistance and growth promotion.

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.

Sequence listing

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Claims

1. A Cladosporium sp strain LJ1 with preservation number of CGMCC NO. 18140.

2. The Cladosporium sp (Cladophiaphora sp.) strain LJ1 according to claim 1, wherein the strain is isolated from the rhizosphere soil of sugarcane.

3. Use of the strain LJ1 of the species verticillium (cladophora sp.) according to any of claims 1 to 2 for the control of blight; the wilt disease is banana wilt disease.

4. Use of the strain LJ1 of the species aspergillus cladosporioides (cladophora sp.) as claimed in any of claims 1 to 2 for promoting the growth of angiosperms; the angiosperm is selected from tomato and/or dendrobium officinale.

5. A method for controlling banana wilt using the strain LJ1 of the genus cladosporium (cladophora sp.) according to any one of claims 1-2, said method comprising: inoculating the strain LJ1 into banana tissue culture seedlings and/or plants; the inoculation method comprises the step of directly inoculating the banana tissue culture seedlings in a culture medium for culturing the LJ1 strain and/or irrigating the activated LJ1 bacterial liquid to the roots of the banana tissue culture seedlings and/or plants.

6. A method of promoting the growth of angiosperms using the strain LJ1 of the genus leptospora (cladophora sp.) according to any one of claims 1 to 2, said method comprising: inoculating the strain LJ1 into a tissue culture seedling and/or a plant of an angiosperm; the inoculation method is that the tissue culture seedling of angiosperm is directly inoculated in a culture medium for culturing LJ1 strain; the angiosperm is selected from tomato and/or dendrobium officinale.