CN111733083A - New endophytic fungus strain PG2 and application thereof - Google Patents
New endophytic fungus strain PG2 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a novel endophytic fungus strain PG2 and application thereof. The new strain PG2 is obtained by trapping and separating from sugarcane, and has a preservation number of CGMCC NO.19647, is a new species of Ramophialophora according to a taxonomic method, has a classification status of Ramophialophora sp, and is selected as a model strain PG 2. Through research, the strain plays a good role in promoting the growth of plants, and the applicant utilizes the strain to co-culture sugarcane and tomatoes and finds that the strain has a good effect in promoting the growth of both the sugarcane and the tomatoes; meanwhile, after inoculation of the rice black-streaked dwarf virus (SRBSDV), the PG2 bacterial liquid is found to obviously reduce the severity of rice diseases and improve the disease resistance of rice to dwarf diseases.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, in particular to a novel endophytic fungus strain PG2 and application thereof.
[ background of the invention ]
The microorganisms have a plurality of beneficial microorganisms which are symbiotic with plants, can effectively promote the growth of crops, simultaneously has a certain disease-resistant effect, can be prepared into non-toxic and pollution-free biological fertilizers and biological pesticides, can reduce the use of chemical fertilizers and pesticides, improve the quality of crops and protect the agricultural ecological environment. At present, in the development and utilization research of beneficial agricultural microorganisms, the applicant has found that a plurality of strains have good effects of promoting growth and resisting diseases simultaneously, for example, Cladophiaphora strains with the application number of 201910884589.9, named as Cladophora LJ1 and application thereof, are separated from soil by the applicant and have double effects of promoting the growth of sugarcane and preventing and treating banana wilt; even some new species, for example, Acidomelaiaccharicola new species named "New species endophytic fungus LZ3 and its application" of patent application No. 201911188968.0, were found, and the strain also had dual effects of promoting growth and preventing and treating banana wilt; the invention relates to an endophytic fungus with the title of 201610652927.2, namely endophytic fungus L-14 for preventing and treating banana vascular wilt and application thereof, which is obtained by trapping and separating from soil by an applicant, belongs to Schizotech, and has the effect of preventing and treating banana vascular wilt; therefore, the growth-promoting bacteria can effectively ensure the growth of plants and improve the disease resistance of the plants, have good compatibility with the environment and can provide a new idea for solving the urgent needs of the current environmental pollution and food safety crisis; meanwhile, as researchers of endophytes, more excellent strains need to be further excavated from abundant and diverse endophyte germplasm resources, so that resource guarantee is provided for promoting the growth of crops.
[ summary of the invention ]
In view of the above, in order to further explore more excellent strains from abundant and diverse endophytic microorganism germplasm resources and provide resource guarantee for promoting crop growth, the invention aims to provide a novel strain Ramophialophora sp.PG2 and application thereof in promoting plant growth and improving disease resistance.
In order to achieve the aim, the invention screens out a new strain Ramophialophora sp.PG2 with the preservation number of CGMCC NO. 19647. Belonging to the kingdom fungi, an unknown species of the genus Ramophialophora. The fungus is preserved in China general microbiological culture Collection center, and the address is as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, is No. CGMCC No.19647 with a preservation date of 2020, 06 and 01 days.
The strain is inoculated on a CMMY culture medium, and after 10 days of culture, the morphological characteristics of observed colonies are shown in FIG. 1 and FIG. 2: morphologically, PG2, like the known species of the genus Ramophialophora, produced a bowknot-like, vial-like spore-forming structure, the conidia of which were formed from a viscous material and released as clusters from the spore-forming orifice. The difference from the kindred species Ramophilarapalogobispora is that: the spore-forming structures of Ramophilalophora globispora are clustered together in a head shape, and the spore-forming structures are short, while the spore-forming structures of PG2 are mostly single-grown and much larger in length and diameter than those of Ramophila globispora.
Further, the strain is obtained by trapping and separating the sugarcane rhizosphere soil.
The invention also comprises the application of the new strain Ramophialophora sp.PG2 in promoting the growth of plants.
Further, the plant is sugarcane and/or tomato.
The invention also discloses the function of the new strain Ramophialophora sp.PG2 in improving the disease resistance of plants to southern black-streaked dwarf.
Further, the plant is rice.
The invention also comprises a method for promoting plant growth by applying the novel strain Ramophialophora sp.PG2, which comprises the following steps: inoculating the strain PG2 into a tissue culture seedling and/or a plant of a plant; the inoculation method is to directly inoculate the tissue culture seedlings of the plants in a culture medium for culturing the PG2 strain.
The invention also comprises a method for improving the disease resistance of rice to southern black-streaked dwarf by applying the new strain Ramophialophora sp.PG2, which comprises the following steps: and (3) irrigating the PG2 bacterial solution cultured by the culture medium to the root system of the rice plant.
Further, the culture medium is a PSB culture medium: 200g of potato, 15g of cane sugar, 1000mL of distilled water and 6 pH.
The invention has the following beneficial effects:
the new strain Ramophialophora sp.PG2 is a new strain discovered by the applicant, can play a good role in promoting the growth of plants, and the applicant utilizes the strain to co-culture sugarcane and tomatoes to discover that the strain has a good role in promoting the growth of the sugarcane and the tomatoes; meanwhile, after inoculation of the rice black-streaked dwarf virus (SRBSDV), the PG2 bacterial liquid is found to reduce the incidence rate of the southern black-streaked dwarf disease of the rice, obviously reduce the incidence severity of the disease of the rice and have the effect of improving the virus resistance of the rice.
[ description of the drawings ]
FIG. 1 is a colony morphology of PG2 strain of the present invention grown in CMMY medium;
FIG. 2 is an enlarged view under a light microscope of the PG2 strain of the present invention;
FIG. 3 is a dendrogram of strain PG2 of the present invention based on ITS phylogeny;
FIG. 4 is a view of the survival condition of the tissue-cultured transplanted sugarcane seedlings after PG2 inoculation;
FIG. 5 is a view of the survival condition of the tissue-cultured transplanted seedlings of sugarcane seedlings without PG2 inoculation;
FIG. 6 shows the growth of sugarcane potted seedlings inoculated with PG 2; in the figure, seedlings of sugarcane inoculated with the PG2 strain are shown on the left, and seedlings of sugarcane not inoculated with the PG2 strain are shown on the right.
FIG. 7 shows the growth of tomatoes inoculated with PG2 under aseptic conditions; in the figure, tomato seedlings inoculated with the strain PG2 are shown on the right, and tomato seedlings not inoculated with the strain PG2 are shown on the right.
FIG. 8 is a graph showing the incidence of rice black-streaked dwarf virus after treatment of PG 2. In the figure, the right side is a rice seedling inoculated with PG2, and the right side is a control rice seedling without inoculation.
[ detailed description ] embodiments
The invention is further illustrated below with reference to the figures and examples and tests.
Example 1:
in this example, a new strain Ramophialophora sp.PG2 with the preservation number of CGMCC NO.19647 was selected. Belonging to the genus Ramophialophora of the kingdom fungi. The fungus is preserved in China general microbiological culture Collection center, and the address is as follows: the No. 3 Xilu Beijing province of Chaoyang, the preservation number is CGMCC No.19647, and the preservation date is 2020, 06 and 01 days.
The strain of the embodiment is separated from sugarcane fields of fruit-melting town dragon villages in Pinguo county of Bai-color city in Guangxi by adopting a trapping method.
The microorganisms of this example were morphologically classified and identified by molecular biology as follows:
1. morphological Classification of strains
The strain is inoculated on a CMMY culture medium, and after 10 days of culture, the morphological characteristics of observed colonies are shown in FIG. 1 and FIG. 2: morphologically, PG2, like the known species of the genus Ramophialophora, produced a bowknot-like, vial-like spore-forming structure, the conidia of which were formed from a viscous material and released as clusters from the spore-forming orifice. The difference from the close-source Ramophilarogispora is that: the spore-forming structures of the Ramophilalophora globispora are gathered together in a head shape, the spore-forming structures are short, most of the spore-forming structures of PG2 are single, and the length and the diameter of the spore-forming structures are much larger than those of the Ramophilalophora globispora.
2. Molecular biological identification
DNA sequence BLAST was performed in GenBank database using the 18S and 28S sequences obtained by sequencing, and the known species having the closest relationship to the strain PG2 was Ramophialophora globispora. The ITS sequences of representative strains of five known species under the genus Ramophilalophora and their kindred species were combined with the ITS sequence of the strain PG2 to construct a phylogenetic tree, as shown in FIG. 3, the results of the phylogenetic tree constructed based on the ITS sequences show that the strain PG2 and Ramophora globispora are clustered in one branch (support rate 100%). In addition, the ITS sequence similarity of PG2 and Ramophialophora globispora model strain LC5696 is only 97%, and based on the identification information of the morphology and the molecular biology, strain PG2 is identified as a suspected new species under Ramophialophora, and is named as Ramophialophora sp.PG2.
Example 2:
the growth promoting effect on the tissue culture transplanted seedlings of the sugarcane is as follows:
(1) planting: filling clean fine sand in a seedling raising tray with 54 holes, and transplanting the acclimatized sugarcane tissue culture seedlings into the seedling raising tray, wherein each hole is provided with one root and one root, and the number of the holes is 3; placing in a light-transmitting greenhouse after planting, and covering with a film for moisturizing. And (5) performing conventional management.
(2) Preparing and inoculating bacterial liquid: the strain is subjected to shake culture for 10 days in a triangular flask containing 150mL of PSB (potato 200g, sucrose 15g, pH6) culture solution and 250mL, then filtered by gauze, mycelia are collected, the culture solution is washed off, water is squeezed out, 7g of mycelia are weighed and smashed by a cooking machine, and 900mL of bacterial solution is prepared; and (5 mL of the seedlings are inoculated at the root irrigation 1d after the sugarcane seedlings are transplanted, and equal amount of water is used as a control.
(3) The subsequent growth condition of the sugarcane seedlings is as follows: and filling the seedling raising trays with 54 holes with fresh red soil, inoculating the seedlings with consistent growth vigor for 35 days, taking the seedlings with one strain in each hole, taking one tray as one repeat, repeating the steps for 3 times, and placing the seedlings on outdoor flat ground after planting.
(4) And (3) investigation: after inoculation, surveying and recording the survival root number of the sugarcane seedlings at 28d, and calculating the survival rate; after the secondary transplanting, 30 plants are taken for investigation at 60d, 10 plants (the best and worst 5 plants are seen by naked eyes) are respectively selected for each repetition, soil is removed, roots are washed clean, plant height is measured, fresh weight is weighed, drying is carried out until constant weight and dry weight are weighed, and test results are shown in table 1:
TABLE 1 influence of PG2 on the survival rate of transplanted tissue culture seedlings of sugarcane and their subsequent growth
| Treatment of | The survival rate is high | Height cm of plant | Fresh weight g | Dry weight g |
| PG2 | 88.9 | 14.3 | 87.8 | 21.6 |
| PG2 | 87.0 | 13.3 | 79.4 | 18.7 |
| PG2 | 77.8 | 19.4 | 147.6 | 34.9 |
| Control (CK) | 70.4 | 13.2 | 71.1 | 16.8 |
As can be seen from Table 1, the average survival rate of sugarcane seedlings inoculated with PG2 strain is 84.57%; the survival rate is improved by 20.1 percent compared with the survival rate of a Control (CK) without inoculating PG2 strain, which shows that PG2 has obvious promotion effect on the survival rate of the transplanted sugarcane tissue culture bottle seedlings. The investigation result after 60d of secondary transplantation shows that the average plant height (15.67cm), the average fresh weight (104.93g) and the average dry weight (25.07g) of the sugarcane seedlings inoculated with the PG2 strain are respectively increased by 18.7%, 47.6% and 49.2% compared with those of non-inoculated controls, which indicates that 1d of inoculation of PG2 after the transplantation of the sugarcane tissue culture seedlings can continuously promote the growth of sugarcane, and the duration is at least 95 d. FIG. 4 is a graph showing the growth of sugarcane seedlings inoculated with the strain PG 2; FIG. 5 is a CK group growth chart of a seedling of uninoculated PG2 sugarcane, and it can be seen from FIGS. 4 to 5 that the sugarcane after inoculation with PG2 has better growth than CK.
Example 3:
the growth promoting effect on the sugarcane pot seedlings is as follows:
(1) planting: mixing the sterilized seedling raising substrate and paddy field soil in a proportion of 1: 13, and filling into a flowerpot (6.5 kg/pot) with the diameter of about 25cm, the lower diameter of about 17cm and the height of 19 cm; transplanting the sugarcane tissue culture seedlings (bay sugar 47) which are cultured in the nutrition cups and have consistent growth vigor into pots, wherein 4 pots are formed in total of 3 plants per pot. After planting, placing the sugarcane in a light-transmitting greenhouse, and covering a film to preserve moisture until the sugarcane recovers growth. And (5) performing conventional management.
(2) Preparing and inoculating bacterial liquid: the strain is subjected to shake culture for 10 days in a triangular flask containing 150mL of PSB (potato 200g, sucrose 15g, distilled water 1000mL, pH6) culture solution and 250mL of culture solution, then filtered by gauze, mycelia are collected, the culture solution is washed off, then squeezed to remove water, 20 g of the culture solution is weighed and smashed, and 600mL of bacterial solution is prepared. After the transplanted sugarcane recovers growth, root irrigation and inoculation are carried out, wherein 150 mL/pot is carried out, and equal amount of water is used as a control. A total of 3 inoculations were performed, each at 10d intervals.
(3) And (3) investigation: taking all plants 100 days after the last inoculation, washing soil at the roots, weighing fresh weight, measuring plant height, drying to constant weight and weighing dry weight, and obtaining test results shown in table 2:
TABLE 2 growth promoting Effect of PG2 on sugarcane under indoor potting conditions
| Treatment of | Height cm of plant | Fresh weight g | Dry weight g |
| PG2 | 109.2 | 769.0 | 400 |
| PG2 | 97.3 | 672.2 | 350 |
| PG2 | 116.2 | 803.8 | 425 |
| PG2 | 114.7 | 787.5 | 400 |
| Control | 103.6 | 547.4 | 285.7 |
As can be seen from Table 2, the sugarcane seedlings inoculated with the PG2 strain had an average plant height (109.35cm), an average fresh weight (758.13g) and an average dry weight (393.8 g); the increase of the content of PG2 in the sugarcane seedlings is 5.6%, 38.5% and 37.8% respectively compared with that of a non-inoculated control, and the inoculation of PG2 under indoor potting conditions can promote the growth of the sugarcane.
FIG. 6 is a growth diagram of potted sugarcane seedlings inoculated with the PG2 strain, wherein the left side of the diagram is the sugarcane seedling inoculated with the PG2 strain, the right side of the diagram is a CK control group, and it can be seen that after the PG2 strain is inoculated, the plant height of the sugarcane seedling is far greater than that of the CK group, which shows that the PG2 strain has a remarkable growth promoting effect on the sugarcane seedling.
Example 4:
growth promoting effect on tomatoes:
(1) culture of PG2 strain: the strain is inoculated on OA (MgSO) by adopting a three-point inoculation method4·7H2O 1.0g，KH2PO41.5g，NaNO31.0g, 10g of oat flour, 11g of agar powder and 1000mL of distilled water) on a culture medium, and connecting 7 dishes together, and culturing in the dark at 25 ℃ for 10 days to form 3 colonies per dish.
(2) Culturing tomato seedlings: sterilizing the surface of tomato seed, accelerating germination, transplanting the germinated seeds to cultured bacterial colonies, inoculating 1 seed to each bacterial colony, placing together with culture dish into sterilized tissue culture bottle, placing at 23 deg.C with illumination intensity of 180 μmol/m2Culturing in an illumination incubator with the illumination time of 16 h/d.
(3) And (3) investigation: after 14d of cultivation, the tomato seedlings are dried to constant weight and then the dry weight is weighed, and the test results are shown in table 3:
TABLE 3 growth promoting Effect of PG2 on tomatoes on plates
| Treatment of | Dry weight g |
| PG2 | 0.019 |
| PG2 | 0.020 |
| PG2 | 0.020 |
| PG2 | 0.027 |
| PG2 | 0.027 |
| PG2 | 0.028 |
| PG2 | 0.017 |
| Control (CK) | 0.018 |
As can be seen from Table 3, the average dry weight of the tomato inoculated with PG2 strain was (0.023 g); the increase of 25.40% compared with the non-inoculated control shows that PG2 has the growth promoting effect on tomatoes.
FIG. 7 is a graph showing the growth of tomato after inoculation with PG2 on a plate, wherein the left side of the graph is a Control (CK) group and the right side is an experimental group inoculated with PG2 strain, and it is evident that tomato seedlings grew better after inoculation with PG2 strain than the control group not inoculated with PG2 strain.
Example 5:
effect of PG2 on rice dwarf resistance:
(1) preparing a bacterial liquid: the strain is subjected to shake culture for 10 days in a triangular flask containing 150mL of PSB (potato 200g, sucrose 15g, distilled water 1000mL, pH6) culture solution and 250mL of culture solution, then filtered by gauze, mycelia are collected, the culture solution is washed off, water is squeezed out, 4g of the culture solution is weighed and smashed, and 120mL of bacterial solution is prepared.
(2) Sowing of rice and inoculation of PG 2: equivalently filling the sterilized seedling culture substrate into transparent plastic cups of (11 multiplied by 15) cm, pouring 40mL of bacterial liquid into each cup, and uniformly stirring; the rice seeds are dibbled and spotted on the substrate in cups after being disinfected, accelerated to germinate and white, 30 grains per cup, and 3 cups in total. And (3) inoculating for the second time when the seedlings grow to 1.5-2.5 leaves, pouring 40mL of bacterial liquid in each cup, and inoculating for 2 times by taking distilled water as a control.
(3) Inoculation of rice black-streaked dwarf virus (SRBSDV): after the second inoculation is finished, placing cup seedlings in a 60-mesh insect prevention net cover, inoculating according to the quantity of the adult white-backed planthopper which is infected by the head virus of 1.0-1.5 per seedling on average, and slightly shaking the plastic cups 1 time in the morning and at night every day to ensure that the rice seedlings are uniformly infected to the greatest extent; after inoculation for 48h, the insects are transferred or released, and the rice seedlings are transplanted into insect-proof net room strip pots. And (5) performing conventional management.
(4) And (3) investigation: and (5) surveying and recording the number of diseased plants 30d after transplanting, calculating the disease incidence, and weighing the fresh weight.
Incidence (%) is the number of diseased plants/total investigated plants × 100%
The results of the above investigation and test are shown in table 4:
TABLE 4 expression of resistance to dwarf after inoculation of rice with PG2
| Treatment of | The incidence of disease% | Fresh weight g |
| PG2 | 94.4 | 21.3 |
| PG2 | 93.3 | 22.7 |
| PG2 | 90.0 | 28.4 |
| Control (CK) | 95.3 | 17.4 |
As can be seen from Table 4, the average incidence of the rice inoculated with the PG2 strain is 92.6%, which is lower than that of the control (95.3%), but the average fresh weight of the plant (24.1g) is increased by 38.7% compared with that of the control (17.4g), which indicates that the effect of reducing the incidence of the rice inoculated with the PG2 strain is not obvious, but the growth of the plant is still better than that of the control, thus obviously reducing the severity of the rice dwarf and improving the disease resistance to the dwarf.
FIG. 8 is a graph showing the growth of rice inoculated with PG2 strain after inoculation with rice black-streaked dwarf virus, wherein the left graph is a Control (CK) group and the right graph is a test group inoculated with PG2 strain, and it is evident that after inoculation with PG2 strain, the rice inoculated with dwarf virus has better growth than the Control (CK) group.
In summary, the Ramophialophora sp.PG2 strain belongs to a new strain, can play a role in promoting the growth of crops and improving the disease resistance of plants to southern black-streaked dwarf, has double functions of promoting the growth and improving the disease resistance of plants, and is a new microbial germplasm resource.
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 (9)
1. The new strain Ramophialophora sp.PG2 has the preservation number of CGMCC NO. 19647.
2. The novel strain Ramophialophora sp.PG2 as claimed in claim 1, wherein said strain is isolated from the soil surrounding the root of sugarcane.
3. Use of the novel strain ramophilophora sp.pg2 as claimed in any of claims 1 to 2 for promoting plant growth.
4. Use according to claim 3, wherein the plant is sugar cane and/or tomato.
5. The use of the novel strain ramophilophora sp.pg2 as claimed in any of claims 1 to 2 for increasing the disease resistance of plants against southern black-streaked dwarf.
6. The use of claim 5, wherein the plant is rice.
7. A method for promoting plant growth using the novel strain ramophilophora sp.pg2 as claimed in any of claims 1 to 2, said method comprising: inoculating the strain PG2 into a tissue culture seedling and/or a plant of a plant; the inoculation method is to directly inoculate the tissue culture seedlings of the plants in a culture medium for culturing the PG2 strain.
8. A method for improving the disease resistance of rice to southern black-streaked dwarf using the novel strain ramophila sp.pg2 as claimed in any of claims 1 to 2, which comprises: and (3) irrigating the PG2 bacterial solution cultured by the culture medium to the root system of the rice plant.
9. The method for improving the disease resistance of the plant to the southern black-streaked dwarf disease by using a novel strain Ramophialophora sp.PG2 as claimed in claim 8, wherein the culture medium is PSB culture medium: 200g of potato, 15g of cane sugar, 1000mL of distilled water and 6 pH.
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| CN112195108A (en) * | 2020-11-09 | 2021-01-08 | 广西壮族自治区农业科学院微生物研究所 | Cladosporium cladosporioides strain MS2 and application thereof |
| CN112625920A (en) * | 2020-12-25 | 2021-04-09 | 广西壮族自治区农业科学院微生物研究所 | Application of disease and insect resistant endophytic fungi |
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| CN112195108B (en) * | 2020-11-09 | 2023-03-14 | 广西壮族自治区农业科学院 | Cladosporium cladosporioides strain MS2 and application thereof |
| CN112625920A (en) * | 2020-12-25 | 2021-04-09 | 广西壮族自治区农业科学院微生物研究所 | Application of disease and insect resistant endophytic fungi |
| CN112625920B (en) * | 2020-12-25 | 2023-04-28 | 广西壮族自治区农业科学院 | Application of disease and insect resistant endophytic fungi |
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