CN107988087B - Blueberry endophytic fungus with growth promoting effect and application thereof - Google Patents
Blueberry endophytic fungus with growth promoting effect and application thereof Download PDFInfo
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Abstract
The invention discloses a blueberry endophytic fungus with a growth promoting effect and application thereof. A blueberry endophytic fungi functional strain 3-41 belongs to Ascomycota (bacterioides) and Pediomycetaceae, and is classically named as DermateaceaePezicula ericae strain 3‑41（Pezicula ericae=Cryptosporiopsis ericae) The strain is preserved in Guangdong province microorganism culture collection center in 2017, 6 and 1, and the preservation number is GDMCC No. 60283. The blueberry endophytic fungi functional strain 3-41 is separated from the root system of wild blueberries, and can infect the root system of the blueberries after being inoculated into potted seedlings of the cultivated blueberry, which mainly invade into the epidermis and cortical cells of the roots, and is a typical endophytic fungi colonization mode. In addition, blueberry pot culture inoculation experiments show that the inoculated strains 3-41 remarkably promote the growth of overground parts of cultivated blueberries, and moreover, the blueberry pot culture can be purely cultured, is easy for industrial production, and has good application prospects.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a blueberry endophytic fungus with a growth promoting effect and application thereof.
Background
Blueberry, the scientific name cowberry fruit, is Vaccinium of Ericaceae (Ericaceae) ((R))Vaccinium) Perennial shrubs are emerging small berry tree species with higher economic value and wide development prospect. Wild blueberries in China are abundant in resources, but for commercially cultivated blueberry varieties, the requirements on soil conditions are strict, the blueberry varieties are favored to be acid (pH is 4.5-5.5) and high organic matter soil, and the cultivation area in China is relatively small. At present, the number of the current day,the blueberry industry in China develops rapidly, commercial cultivation areas move to the south continuously, and besides main old production areas such as Changbai mountain, Liaodong peninsula, Jiaodong peninsula, Yangtze river basin, southwest production area and the like, the blueberry cultivation (southern highbush, rabbit eye blueberry) in south China such as Guangdong, Guangxi, Fujian and the like develops rapidly. However, the temperature of a new cultivation area in south represented by Guangdong province is high, soil nutrients are deficient, and the development of the blueberry industry is severely restricted.
Endophytic fungi (Endophytic fungi) are those which live in healthy plant tissue at some or all of their life cycle and which are infected by the host plant without causing significant disease symptoms. Among the endophytic fungi, mycorrhizal fungi are an important group, and the root systems of more than 90% of terrestrial plants in nature can establish a reciprocal symbiotic relationship with the endophytic fungi. In recent years, mycorrhizal technology is increasingly applied to agriculture and forestry, and the physiological and ecological functions of endophytic fungi are obvious and proved to be capable of promoting plant growth, improving plant resistance and increasing the success rate of introduction.
Therefore, the mycorrhizal technology is a good way to improve the blueberry cultivation and planting conditions in south China, but at present, the bacterial strains which have high-efficiency promotion effects on the growth of the cultivated blueberries are seriously deficient, and the application of related high-efficiency bacterial strains in blueberry cultivation is rarely reported. Therefore, the method has important significance for promoting the development of the blueberry industry in China and building an environment-friendly society in the future by screening the high-efficiency blueberry endophytic fungi to improve the cultivation and planting conditions of the blueberries.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and separates a blueberry endophytic fungus strain 3-41 with high-efficiency growth promoting effect from a wild blueberry root system, wherein the strain can infect the blueberry root system and mainly invade into root epidermis and cortical cells; but also can obviously promote the growth of the overground part of the cultivated blueberry.
The first purpose of the invention is to provide a blueberry endophytic fungi functional strain 3-41.
The second purpose of the invention is to provide the application of the functional strain 3-41 of the endophytic fungi of blueberries in blueberry cultivation.
The third purpose of the invention is to provide a blueberry growth promoter.
The fourth purpose of the invention is to provide a growth promoting fertilizer suitable for blueberries.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a blueberry endophytic fungi functional strain 3-41 belongs to Ascomycota (bacterioides) and Pediomycetaceae, and is classically named as DermateaceaePezicula ericae strain 3-41（Pezicula ericae = Cryptosporiopsis ericae) The strain is preserved in Guangdong province microbial strain collection center with the preservation date of 2017, 6 and 1 and the preservation number of GDMCC number 60283.
The strain is observed by the morphology and the molecular means of pure culture of the fungus and the root infection form of the inoculated blueberries, and the three methods are jointly identified, so that the filamentous fungus does not produce spores, and hyphae are separated; on a PDA flat plate, the colony morphology is irregular, flat, spotty and compact, the color is light brown, and the edge is white; the strain grows slowly under pure culture conditions, the optimal growth temperature is 24 ℃, and the optimal growth pH is 7.0.
The strain can infect blueberry root systems and mainly invade into root epidermis and cortical cells; but also can obviously promote the growth of the overground part of the cultivated blueberry.
Therefore, the application of the blueberry endophytic fungi functional strain 3-41 in blueberry cultivation also belongs to the protection scope of the invention.
Preferably, the application is to promote growth of the overground part of the blueberries. .
Preferably, the application is to increase the amount of growth.
Compared with the prior art, the invention has the following beneficial effects:
the blueberry endophytic fungi functional strain 3-41 is separated from the root system of wild blueberries, and can infect the root system of the blueberries after being inoculated into potted seedlings of the cultivated blueberry, which mainly invade into the epidermis and cortical cells of the roots, and is a typical endophytic fungi colonization mode. In addition, blueberry pot culture inoculation experiments show that the inoculated strains 3-41 remarkably promote the growth of overground parts of cultivated blueberries, and moreover, the blueberry pot culture can be purely cultured, is easy for industrial production, and has good application prospects.
Drawings
FIG. 1 shows the inoculum strainPezicula ericaePhotos of hypha in cells of the epidermis and cortex of a blueberry root system infected by strain 3-41.
FIG. 2 shows the strains of the present inventionPezicula ericaePhotographs of the colony morphology of strain 3-41.
FIG. 3 shows the inoculum strainPezicula ericaeAnd comparing the biomass of the overground part and the underground part of the blueberry plant after 6 months of culture of strain 3-41 and an uninoculated control.
FIG. 4 shows the inoculum strainPezicula ericaeAnd comparing the total biomass of the blueberry plants after 6 months of culture of strain 3-41 and an uninoculated control.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1: separation and screening of blueberry endophytic fungus strains 3-41
1. Strain isolation
The sampling place is the southwest area of China, Lijiang City, Yulong Naxi autonomous county, Ludian county and Sanjiacun in Yunnan province. The type of the land is a mountain land type, the type of the soil is forest soil, the geographical range is N26 degrees 45 '58' to N27 degrees 14 '45', E99 degrees 34 '37' to E100 degrees 17 '46', and the altitude range is 2016 m to 2647 m. The collected sample is wild blueberry, Yunan cowberry fruit (A)V.duclouxii(Lev.l) hand. -Mazz.) and crow fruit ((Lev.l)V.fragileFranc-h.) with soil root system.
After being brought back to the laboratory, the root sample was washed with tap water for 1H and then with 30% H in a clean bench2O2Surface sterilization is carried out for 5-7 minutes, and thenCleaning the hairy roots with the fungus water for 2 times, and cutting the hairy roots into root sections with the length of 1-2 mm; placing the root segments in sufficient PDA (containing 0.03 g.L)-1Streptomycin) plates, 5 root segments per dish, and dark culture in an incubator at 25 ℃. Each plant was cultured in 15 dishes for root-like culture. After culturing for 2-4 weeks, after the bacterial colony grows out of the root section, picking hyphae at the edge of the bacterial colony to a new PDA plate by using a picking needle for purification culture until the bacterial colony is single.
2. Strain screening
And (3) screening strains by utilizing pure culture mycorrhiza to synthesize and identify:
(1) test plants: the plant for pure culture of mycorrhiza resynthesis is obtained by a plant tissue culture method, the variety is 'blue rain' of southern highbush blueberries, and the plant is used as a test plant after a rooting seedling is obtained by induction.
(2) Liquid microbial inoculum of strains 3-41: the culture medium is liquid PDA culture medium, inoculating strain 3-41 cultured on flat plate for 4 weeks into triangular flask, placing on shaking table for shaking, controlling temperature at 25 deg.C and 150 r.min-1Shake for 2 weeks.
(3) The pure culture mycorrhiza synthesis test process comprises the following steps: inoculating the shaken bacterial liquid 3-41 into the rooted seedlings in the bottle on an ultraclean workbench, wherein the volume ratio of the bacterial liquid to the blueberry tissue culture medium is 1:3, inoculating 5 blueberry seedlings together, and culturing for 2 weeks at 25 ℃ after inoculation.
(4) And (3) detecting the root system of blueberry infected by the strain 3-41:
staining was performed by modified trypan blue staining method, and observation was made by optical microscope whether the root segment was infected with hyphae and whether there was a generation of a loop of hyphae in the infected root epidermal cells. The specific process is as follows:
1) material taking: taking out the seedling from the culture bottle, cutting off the root system, washing, and cutting into root segments of about l cm.
2) Fixing: the root segments are fixed in FAA fixing solution and can be stored for a long time at a temperature of about 10 ℃ (per 100 mL FAA fixing solution formula: 5 mL of 38% formaldehyde solution, 5 mL of acetic acid and 90 mL of 50% alcohol).
3) And (3) transparent treatment: treating with 5% KOH, fixing, and treating in 60 deg.C water bath for 30 min. After cleaning the root sample with clear water, immersing the root sample in 1% HCl for 3-4 min for acidification treatment, or cleaning the root sample with distilled water without acidification, and directly dyeing.
4) Trypan blue staining: the above-mentioned treated roots were put into 0.2% trypan blue staining solution (per 1000 mL of 0.2% formula of trypan blue staining solution: 300 mL of lactic acid +300 mL of glycerin +400 mL of water +2.15 g of trypan blue + 350 g of phenol), and the mixture was heated in a water bath to 60 ℃ and maintained at a constant temperature for 30 min.
5) And (3) decoloring: taking out the dyed root sample, sucking dry the surface dye solution, and then putting the root sample into lactic acid glycerol for soaking overnight, wherein the volume ratio of the lactic acid glycerol to the lactic acid glycerol is 1: 1.
6) Tabletting: dropping a drop of glycerol on a clean glass slide, taking out the decolored root end, putting the root end on the glass slide (30-40 roots) by using tweezers, covering a cover plate, and lightly pressing to spread the roots and extrude bubbles.
7) Microscopic examination: selecting 60 root segments with the length of about 1 cm, and observing whether the root segments are infected or not and whether aseptic silks are colonized in infected root epidermis and cortex cells.
3. Results
According to the pure culture mycorrhiza resynthesis test result, a strong endophytic fungus hypha which can infect the root system of the blueberry and can be formed in epidermal and cortical cells of the root system of the blueberry is screened, and a better strain is selected and named as a strain 3-41 (figure 1).
Example 2: identification of blueberry endophytic fungus strain 3-41
1. Morphological identification
The hypha of the strain has septation and does not produce spores; the colony morphology on the PDA plate was irregular, flat, spotty and dense, with a light brown color and white edges (FIG. 2).
2. Molecular identification
In the research, a Tiangen plant genome DNA extraction kit is selected to extract the fungal genome DNA; ITS segments of the fungal rRNA gene were amplified using ITS1 and ITS4 primers. And sequencing the amplified ITS segment.
The full length of the internal transcribed spacer sequence of the fungal ribosome deoxyribonucleic acid of the strain is shown as SEQ ID NO: 1:
ACCTGCGGAGGGATCATTACAGAGACTCTGCCCTTTGGGTAGACCTCCCACCCTGTGTCGTTATACCTTCGTTGCTTTGGCGGGCCGCGGGGCCCCGGCCCCGCCCCTGGCTCCGGCTAGGGCGCGCCCGCCAGAGGACCTCAAAACCTGAATGTTAGTGTCGTCTGAGTACTATATAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTACAACCCTCAAGCTCTGCTTGGTCTTGGGCGTCACCGGTCCCGGTGTGCCTTAAAATCAGTGGCGGCGCCATCTGGCTCTAAGCGTAGTACATACTCTCGCTACAGACGTCCGGTGGATGCTGGCCAGCAACCCCCAACTTATCAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAA
ITS sequences were aligned in NCBI database and found that ITS rRNA gene sequences of strains 3-41 were found to be homologous to species of the genus AphelosporaPezicula ericae The strain CBS 120290 has the highest similarity (100%).
The results of the identification of the strains 3-41 show that the strains belong to the phylum Ascomycota (bacterioides) and the family Pediomycetaceae (Dermateiaceae), and the taxonomic name of the strains isPezicula ericae strain 3-41（Pezicula ericae = Cryptosporiopsis ericae) The strain is preserved in Guangdong province microbial strain collection center in 2017, 6 and 1, and the preservation number is GDMCC number 60283.
Example 3: research on inoculation effect of blueberry endophytic fungus strain 3-41 on blueberry potted seedlings
1. Test plant
The blue rain blueberry (Bluranin) tissue culture seedlings are domesticated on a moss substrate and cultured for 8 months (2015.11-2016.06).
2. Preparation of liquid bacterial agent of strain 3-41
Selecting PDA plates (dark culture at 25 ℃ for 4 weeks) of the strains 3-41, selecting hyphae with vigorous growth on the edges of the colonies, and inoculating the hyphae into a triangular flask containing 70 ml of PDA culture solution. Wrapping with black cloth on a shaker at 25 deg.C for 120 r min-1Cultured for 2 weeks.
3. Inoculation method
By adopting a root dipping method, the strains 3-41 are filamentous fungi, and the shake fermentation hyphae are easy to be aggregated into large spherical fungus clusters, so that the fungus clusters are broken by treating for 30 min with a magnetic stirrer before inoculation and then are dipped with roots.
The test culture medium is a mixed medium (volume ratio is 1:1: 2) of red soil, moss and turf, and is sterilized at high temperature and high pressure for two times at 121 ℃ for 1 h. Each treatment was repeated 4 times, with no inoculation control, water management of acidified water pH =4.5, fertilization management of 20ml nutrient solution per half month (formulation: N: P: K =4: 1:2, N1.34 g. L)-1Trace elements and iron salts were identical to 1/2 WPM medium) were harvested after 6 months of culture (2016.12.10). After the samples are collected, the fresh weight and the total biomass of the overground and underground parts of the plants are detected, and the method for observing the infection condition of the inoculated blueberry seedlings is the same as the method for detecting the blueberry root system infected by the strains 3-41 in the embodiment 2.
4. Results
Compared with a control, the inoculated strain 3-41 has a promoting effect on the overground part growth of the blueberry plant (figures 3 and 4), wherein the overground part growth is improved by 20 percent and reaches a remarkable level, and the total growth amount of the plant is also obviously promoted by 8.02 percent. Tests show that the strains 3-41 show higher growth promotion effect on blueberry growth, and have good application prospect and market value.
Claims (5)
1. Blue rain blueberry endophytic fungus functional strainPezicula ericae 3-41, which is deposited at the Guangdong province culture Collection center on 6 th and 1 st in 2017, with the deposit number GDMCC number 60283.
2. "blue rain" blueberry endophytic fungi functional strain according to claim 1Pezicula ericae 3-41 in the cultivation of blueberries in the blue rain, which is characterized in that the application is to promote the growth of the overground parts of the blueberries in the blue rain.
3. "blue rain" blueberry endophytic fungi functional strain according to claim 1Pezicula ericae 3-41 in the cultivation of blueberries in the blue rain, which is characterized in that the application is to improve the growth amount of blueberries in the blue rain.
4. A blue rain blueberry growth promoter is characterized by comprising the blue rain blueberry endophytic fungi functional strain in claim 1Pezicula ericae 3-41。
5. A growth promoting fertilizer suitable for blue rain blueberries, which is characterized by comprising the blue rain blueberry endophytic fungi functional strain in claim 1Pezicula ericae 3-41 and organic fertilizer.
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| CN110511878B (en) * | 2019-07-31 | 2021-06-11 | 中国林业科学研究院亚热带林业研究所 | Novel aschersonia with bacteriostatic ability and lignocellulose degradation activity and application thereof |
| CN111187724B (en) * | 2020-03-02 | 2022-02-01 | 安庆师范大学 | Endophytic fungus with dark blueberry root and application thereof |
| CN112790107A (en) * | 2021-01-18 | 2021-05-14 | 安徽师范大学 | Method for promoting growth of blueberry seedlings |
| CN116640670B (en) * | 2023-05-12 | 2024-05-24 | 华南农业大学 | Purple flower and sycamore endophytic fungus with growth promoting effect and application thereof |
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| CN104004666A (en) * | 2014-06-05 | 2014-08-27 | 福建农林大学 | Endophytic fungi capable of promoting China fir growth |
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