CN112522116B

CN112522116B - Fungus for producing fragrance in roots of Bidens parviflora and application

Info

Publication number: CN112522116B
Application number: CN202011536894.8A
Authority: CN
Inventors: 田凡; 廖小锋; 李从瑞; 罗在柒; 颜凤霞; 王莲辉; 姜运力; 罗红
Original assignee: GUIZHOU ACADEMY OF FORESTRY
Current assignee: GUIZHOU ACADEMY OF FORESTRY
Priority date: 2020-12-23
Filing date: 2020-12-23
Publication date: 2022-02-01
Anticipated expiration: 2040-12-23
Also published as: CN112522116A

Abstract

The invention discloses a group of fungus producing aroma in the root of the Bidens parviflora and application thereof, which are named Tulasnella. sp XYDL003 and Tulasnella. sp XYDL040, wherein the two strains are preserved in China center for type culture Collection (CCTCC for short, Wuhan city Wuchang Loa Jia shan, Zi-codling: 430072) in 5-15 months in 2020, and the preservation numbers are CCTCC M2020122 and CCTCC M2020123 respectively. The aroma-producing fungi is separated from roots of paphiopedilum micranthum and has strong fruit aroma, in aroma metabolites produced by the aroma-producing fungi, the gamma-decalactone has strong coconut and peach aroma, and the discovery of the fungus provides a new microbial resource for replacing plants to produce essences and fragrances originally.

Description

Fungus for producing fragrance in roots of Bidens parviflora and application

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a group of fungi capable of producing fragrance in blattbulume orchioides root and application thereof.

Background

Paphiopedilum (Paphiopedilum barbargeum Tang et Wang) belongs to the Orchidaceae (Orchidaceae) Paphiopedilum (Paphiopedilum) perennial herbaceous plant, is listed in the red name of IUCN species, belongs to IUCN endangered species, and is mainly distributed in the land or rock space of the cryptic polylithic limestone hills with the altitude of 800-. Distribution of paphiopedilum lobule in Guizhou is also extremely rare and belongs to small population distribution. Many studies have shown that the distribution and specificity of mycorrhizal fungi affects the distribution of orchids, leading to the rarity of orchids, which has been widely discussed.

The sources of the flavors and fragrances mainly comprise two modes of natural and chemical synthesis. Although the period of the essence and flavor in the chemical synthesis path is short, the method has the defects of poor reaction selectivity, more byproducts, environmental pollution and the like; although natural perfumes derived from aromatic plants and animal musk are high in quality, the raw materials are limited, the cost is high, the yield is low, and the market demand cannot be met. The aroma-producing microorganism utilizes natural raw materials to synthesize a large amount of natural aroma substances, and has the advantages of short period, high quality, safety, environmental protection and the like. Since the microbial aroma production is reported for the first time in 1923, the microbial aroma production technology has become a research hotspot for the development of flavors and fragrances, and at present, the reports of aroma-producing microorganisms are mostly fungi.

The endophyte is taken as a special microbial resource, is widely distributed in plants, and is co-evolved with host plants, so that abundant and various metabolites with biological activity can be produced. Therefore, the method has extremely important significance in the development of flavor and fragrance products by screening and separating endophyte resources and utilizing biotechnology. At present, few reports are provided for separating and screening aroma-producing endophytic fungi from paphiopedilum micranthum.

Therefore, it is necessary to develop a novel aroma-producing microorganism.

Disclosure of Invention

In view of the above, the present invention provides a group of fungi for producing aroma in the root of Bidens lobular and applications thereof.

In order to solve the technical problems, the invention discloses a group of aroma-producing fungi in the root of the Kappaphycus parvifolius, which are named Tulasnella.sp XYDL003 and Tulasnella.sp XYDL040, wherein the two strains are preserved in China center for type culture Collection (CCTCC for short, Wuhan city Wuchang Lojia mountain, postal code: 430072) in 5-15 th 2020 with the preservation numbers of CCTCC M2020122 and CCTCC M2020123 respectively.

Sp XYDL040, which is a fungus producing perfume in the roots of blattbulume, is optionally characterized as follows: the partition is a barrel hole diaphragm, and the barrel hole covering pad is not provided with a through hole and is arc-shaped; on a PDA culture medium, the colony is milky white at the early stage, grows by adhering to the culture medium, and aerial hyphae are undeveloped; the later stage is light brown, the edge is velvet-shaped, irregular and jagged, and aerial hyphae are developed and protrude; the growth rate of hyphae is 0.10-0.13mm/h, the hyphae branches at right angles or nearly at right angles, the hyphae and the candida cells are accumulated into piles, the candida cells are round or oval, the size is 6.79-16.39 multiplied by 11.63-45.68 microns, the chain length of the candida cells is generally 4-5 cells, the hyphae have the diameter of 4.39-10.52 microns, the cells have the length of 61.62-126.21 microns, and the distance between the first partition of the branch and the branch point is 2.23-9.15 microns.

The aroma-producing fungus Tulasnella. sp XYDL003 in the root of Bidens parviflora is characterized as follows: the partition is a bung hole diaphragm, the bung hole covering pad has no perforation, and the bung hole covering pad is straight; on a PDA culture medium, the colony is light yellow in early stage, grows by attaching to the culture medium, and has a smooth surface; the later colony is milky white, aerial hyphae are developed, the edge is neat, the thickness is uniform, and the later colony is in a velvet shape; the growth speed of hyphae is 0.08-0.14mm/h, the hyphae branches at right angles or nearly at right angles, the hyphae and the candida cells are accumulated into piles, the candida cells are circular or elliptical, the size is 8.46-10.23 multiplied by 18.37-37.36 microns, the chain length of the candida cells is generally 5 cells, the hyphae have the diameter of 2.39-6.01 microns, the cells have the length of 57.92-135.72 microns, and the distance between the first partition of the branch and the branch point is 2.07-8.73 microns.

Alternatively, the fungus is isolated from the paphiopedilum microphyllum root system.

The invention also discloses an application of the fungus for producing fragrance in the blattbulume root in preparing essence and spice.

Sp XYDL040 volatile substance, a fragrance-producing fungus in the root of blattbulume, is composed as follows: gamma decalactone 78.57%; 2.90 percent of ethanol; 0.32 percent of 2, 3-butanedione; 0.23 percent of 3-methyl-3-butene-1-ol; 0.30% of hexanal; 3.05% of trans-alpha-bergamotene; 0.73% of beta-bisabolene; other undetermined 8.83%;

the volatile substance of the aroma-producing fungus Tulasnella sp XYDL003 in the root of the Bidens parviflora comprises the following components: 96.79% of propyl decalactone, 0.78% of ethanol, 0.24% of 2, 3-butanedione, 0.16% of n-hexanal, 0.17% of benzaldehyde, 0.21% of trans-alpha-bergamotene and 0.17% of beta-bisabolene.

Alternatively, the major volatile substances of the aroma-producing fungi Tulasnella. sp XYDL003 and Tulasnella. sp XYDL040 in the roots of Bidens parviflora are valactone.

Compared with the prior art, the invention can obtain the following technical effects:

1) the method carries out morphological and molecular identification on the Mucuna fungus producing strong fruit fragrance, and utilizes an HP6890/5975C gas-phase-mass spectrometer to identify the volatile substance composition of the Mucuna fungus.

2) The endophytic fungi strains Tulasnella. sp XYDL040 and Tulasnella. sp XYDL003 can synthesize lactone volatile aromatic substances mainly containing gamma decalactone, have elegant aroma, are suitable for blending various essences, and are generally applied to the aroma industry. Thus, it is demonstrated that the strains tulasella.sp XYDL040 and tulasella.sp XYDL003 have a greater development potential in the production of fragrance raw materials.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a colony morphology map of the strains Tulasnella. sp XYDL040 and Tulasnella. sp XYDL003 of the present invention; wherein a represents Tulasnella. sp XYDL040, and b represents Tulasnella. sp XYDL 003;

FIG. 2 shows hyphae and moniliform cells of strains Tulasnella. sp XYDL040 and Tulasnella. sp XYDL003 according to the invention; wherein a represents Tulasnella. sp XYDL040, and b represents Tulasnella. sp XYDL 003;

FIG. 3 is a diagram of the ultrastructure of membranes of the strains Tulasnella. sp XYDL040 and Tulasnella. sp XYDL003 according to the invention; wherein a represents Tulasnella. sp XYDL040, and b represents Tulasnella. sp XYDL 003;

fig. 4 is a total ion flow diagram of volatile aroma components of strain tulasnella. sp XYDL040 of the present invention;

fig. 5 is a total ion flow diagram of volatile aroma components of strain tulasnella. sp XYDL003 of the present invention.

Detailed Description

The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.

EXAMPLE 1 isolation of endophytes

1. Isolation of bacteria

Selecting a root sample of 2-3 years old healthy paphiopedilum, washing for 3-5h in running water state by tap water after soaking for 2h by rhizome soap water, slightly scraping off root hair, root quilt and other accessories on a super clean workbench by using a scalpel, performing microscopic examination under a microscope, selecting a root segment with a mycelial cluster, placing the root segment in a culture dish, and washing for 3-5 times by using sterile water; sterilizing with 75% ethanol for 1min, sterilizing with 0.1% mercuric chloride solution for 4-5min, and washing with sterile water for 5 times; cutting the sterilized root segment into 2-3cm segments, subpackaging in a culture dish with sterile water and diameter of 6cm, and gently scraping the root segment with a scalpel and a pair of tweezers to free the mycelial clusters in the cortex of the root segment; culturing at 24 deg.C, observing under microscope after 24 hr, sucking germinated mycelium with pipette in dark field of microscope, transferring to PDA culture medium, culturing at 24 deg.C for 48 hr, examining, selecting germinated mycelium on the culture medium, culturing in PDA culture medium, purifying, and storing strain in inclined plane of test tube.

2. Cultivation of the Strain

And (3) after the endophytic strains obtained by separation are subjected to aroma smelling evaluation and screening, inoculating the strains XYDL040 and XYDL003 on a solid PDA culture medium, and culturing at the constant temperature of 28 ℃ by illumination. The PDA culture medium formula is as follows: 15g of agar, 20g of glucose, 200g of potatoes and 1L of distilled water. Cutting potato into strips or blocks, adding distilled water, boiling for 30min, adding glucose and agar, stirring, adding water, diluting to constant volume of 1L, subpackaging in conical flask, sterilizing for 20min, taking out, pouring into 9cm culture dish, and cooling.

Example 2 identification of endophytes

(I) morphological identification

1. Identification method

a. Microscopic structural features of bacterial colony and hypha

Inoculating the strains XYDL040 and XYDL003 in the middle of a PDA culture medium plate, clamping a sterile cover glass by using a small forceps, and obliquely inserting the cover glass into the culture medium at an angle of 45 degrees at a certain distance from an inoculation point; when the hyphae grew to about 2/5 degrees on the cover slip, the cover slip was removed and the hyphal structural features were observed under a microscope.

b. Diaphragm ultrastructure

1) Material drawing and fixing: discarding the culture solution, adding electron microscope stationary liquid, fixing at 4 deg.C for 2-4h, centrifuging at low speed until cell pellet with mung bean size can be seen at the bottom of tube, wrapping with 1% agarose, and rinsing with 0.1M phosphate buffer PB (pH7.4) for 3 times, each for 15 min.

2) Post-fixing: 1% osmic acid 0.1M phosphate buffer PB (pH7.4) was fixed at room temperature (20 ℃) for 2 h; 0.1M phosphate buffer PB (pH7.4) was rinsed 3 times for 15min each.

3) And (3) dehydrating: the tissue is sequentially added with 50% -70% -80% -90% -95% -100% -100% alcohol-100% acetone for upward dehydration for 15min each time.

4) And (3) infiltration: acetone 812 embedding medium 1: 12-4h, acetone 812 embedding medium 2: 1 overnight, pure 812 embedding medium 5-8h, pouring the pure 812 embedding medium into an embedding plate, inserting the sample into the embedding plate, and then, baking at 37 ℃ overnight.

5) Embedding: polymerizing for 48h in an oven at 60 ℃.

6) Slicing: and (5) slicing the ultrathin slice with the ultrathin slicer to obtain a 60-80nm ultrathin slice.

7) Dyeing: uranium lead double staining (2% uranium acetate saturated alcoholic solution, lead citrate, each staining for 15min), slicing and drying overnight at room temperature. Observing under a transmission electron microscope, and collecting and analyzing images.

2. Identification results

The strain XYDL040 has the main characteristics that: the partition is a barrel hole diaphragm, and the barrel hole covering pad is not provided with a through hole and is arc-shaped; on a PDA culture medium, the colony is milky white at the early stage, grows by adhering to the culture medium, and aerial hyphae are undeveloped; the later stage is light brown, the edge is velvet-shaped, irregular and jagged, and aerial hyphae are developed and protrude; the growth rate of hyphae is 0.10-0.13mm/h, the hyphae branches at right angles or nearly at right angles, the hyphae and the candida cells are accumulated into piles, the candida cells are round or oval, the size is 6.79-16.39 multiplied by 11.63-45.68 microns, the chain length of the candida cells is generally 4-5 cells, the hyphae have the diameter of 4.39-10.52 microns, the cells have the length of 61.62-126.21 microns, and the distance between the first partition of the branch and the branch point is 2.23-9.15 microns. See fig. 1a, fig. 2a and fig. 3 a.

The strain XYDL003 has the main characteristics that: the partition is a bung hole diaphragm, the bung hole covering pad has no perforation, and the bung hole covering pad is straight; on a PDA culture medium, the colony is light yellow in early stage, grows by attaching to the culture medium, and has a smooth surface; the later colony is milky white, aerial hyphae are developed, the edge is neat, the thickness is uniform, and the later colony is in a velvet shape; the growth speed of hyphae is 0.08-0.14mm/h, the hyphae branches at right angles or nearly at right angles, the hyphae and the candida cells are accumulated into piles, the candida cells are circular or elliptical, the size is 8.46-10.23 multiplied by 18.37-37.36 microns, the chain length of the candida cells is generally 5 cells, the hyphae have the diameter of 2.39-6.01 microns, the cells have the length of 57.92-135.72 microns, and the distance between the first partition of the branch and the branch point is 2.07-8.73 microns. See fig. 1b, fig. 2b and fig. 3 b.

(II) molecular characterization

1. Extracting strain DNA:

1.1, sucking 300 mul of bacterial liquid, transferring the bacterial liquid to a 1.5ml centrifuge tube, adding 8 mul of lysozyme into the tube, fully shaking and uniformly mixing, and performing enzymolysis for 30min at room temperature.

1.2, adding 300 mu l of gelatin Solution and uniformly mixing; adding 4 μ l RNase A, mixing, and keeping the temperature at 55 deg.C for 10 min; then 4. mu.l of protease K was added and the mixture was incubated at 55 ℃ for 30 min.

1.3, if the viscosity of the obtained lysate is low, 300. mu.l of PB Solution is directly added, the mixture is fully shaken and uniformly mixed, the mixture is centrifuged at 12,000rpm at room temperature for 5min, and the supernatant is completely transferred to a GenClean column sleeved in a 2ml collection tube.

Centrifuging at room temperature of 1.4 and 8,000rpm for 1min, taking down GenClean column, and discarding waste liquid in collection tube.

1.5, place GenClean column back into the collection tube, add 500. mu.l Wash Solution, 8,000rpm, centrifuge for 1min at room temperature. The GenClean column was removed and the waste stream from the collection tube was discarded.

1.6, repeating the step 1.5 once.

1.7, place the GenClean column back into the collection tube, centrifuge at 12,000rpm for 1min at room temperature to remove residual Wash Solution.

1.8, put GenClean column into a new clean 1.5ml centrifuge tube, add 50. mu.l of Elution Buffer to the center of GenClean column, put into 37 ℃ oven for 2 min.

1.9, 12,000rpm, and centrifuged at room temperature for 1 min.

TABLE 1 primer sequences

And (3) PCR amplification: 30 μ L system

TABLE 2 PCR System (30. mu.l)

10*Taq Buffer	3μl
		dNTP	1μl
Enzyme	1μl
		H₂O	21μl
Upstream primer (10. mu.M)	1μl
		Downstream primer (10. mu.M)	1μl
Form panel	2μl

Reaction procedure:

TABLE 3 reaction procedure

95℃	10min	1
			94℃	30s	35cycles
55℃	30s
			72℃	1min
72℃	5min	1

2. Gel electrophoresis detection

After the reaction, 5. mu.l of PCR product was taken, detected by 1% agarose gel electrophoresis, and photographed in a SYNGENE GBOX gel imaging system using 100bp DNA ladder Marker, GV staining solution.

3. Sequencing and comparison of PCR products

Qualified PCR products were sequenced. Sequencing results are compared through BLAST by utilizing GeneBank, and the mycorrhizal fungi species are determined by combining morphological observation results.

The rDNA-ITS sequences of the XYDL040 and XYDL003 strains were submitted to the NCBI database and aligned using the BLAST tool.

4. Sequencing results of rDNA-ITS segment of strain

Nucleotide sequences of the XYDL003 and XYDL040 strains are respectively shown in SEQ ID No.3 and SEQ ID No.4, and rDNA-ITS sequences of the XYDL040 and XYDL003 strains are submitted to an NCBI database, and sequence alignment is performed by using a BLAST tool. The results give: the similarity of the amplified fragment of the strain XYDL040 and the Tulasnella sp gene JN253524 published in GeneBank reaches 99.46%. Thus, the strain was identified from morphological characteristics and molecular level as a Tulasnella sp. The similarity of the amplified fragment of the strain XYDL040 and the Tulasnella sp gene MH348614 published in GeneBank reaches 99.42%. Thus, the strain was identified as a Tulasnella sp.

Based on the above characteristics, strains XYDL040 and XYDL003 were identified as Tulasnella sp. The two strains are preserved in China Center for Type Culture Collection (CCTCC for short, Wuhan city, Wuchang Lodojia mountain, zip code: 430072) in 5 months and 15 days in 2020, with the preservation numbers of CCTCC M2020123 and CCTCC M2020122 respectively.

Example 2 aroma component extraction

1. Extracting aroma components of strains XYDL040 and XYDL 003:

taking 1.5g of a uniform mixing sample, placing the uniform mixing sample in a sampling bottle of a 50mL solid phase micro-extraction instrument, inserting a manual sample injector provided with a 2cm-50/30 mu m DVB/CAR/PDMS stablefex fiber head, extracting for 50min in a headspace manner under a flat plate heating condition at 60 ℃, then removing the extraction head, immediately inserting the extraction head into a sample inlet (the temperature is 250 ℃) of a gas chromatograph, and carrying out thermal desorption for 6min for sample injection.

2. GC-MS analysis of volatile substances of strains XYDL040 and XYDL003

The chromatographic column is HP-5MS (60m × 0.25mm × 0.25 μm) elastic quartz capillary columnWarming to 41 deg.C (keeping for 2min), and heating to 3 deg.C/min^-1Heating to 176 deg.C, and heating at 8 deg.C/min^-1Heating to 304 deg.C for 63 min; the temperature of the vaporization chamber is 250 ℃; the carrier gas is high-purity He (99.999%); column front pressure 16.11psi, carrier gas flow 1.0mL min^-1(ii) a No shunt sampling; solvent delay time: 3 min.

The ion source is an EI source; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; electron energy 70 eV; emission current 34.6 μ A; multiplier voltage 1718V; the interface temperature is 280 ℃; the mass range is 29-500 amu. And searching and checking the Nist 14 and Wiley275 standard mass spectrograms of each peak in the total ion flow graph by a mass spectrum computer data system, determining X volatile chemical components, and measuring the relative mass fraction of each chemical component by using a peak area normalization method.

3. GC-MS analysis results of volatile substances of strains XYDL040 and XYDL003

As shown in fig. 4 and 5, the main volatile substances of the aroma-producing fungi in the roots of the blattbulume are as follows:

sp XYDL 040: gamma decalactone 78.57%; 2.90 percent of ethanol; 0.32 percent of 2, 3-butanedione; 0.23 percent of 3-methyl-3-butene-1-ol; 0.30% of hexanal; 3.05% of trans-alpha-bergamotene; 0.73% of beta-bisabolene; others were not defined 8.83%.

Sp XYDL 003: 96.79% of propyl decalactone, 0.78% of ethanol, 0.24% of 2, 3-butanedione, 0.16% of n-hexanal, 0.17% of benzaldehyde, 0.21% of trans-alpha-bergamotene and 0.17% of beta-bisabolene.

In the produced aroma metabolites, the gamma-decalactone has strong coconut and peach fruity aroma, and the discovery of the gamma-decalactone provides a new microbial resource for replacing the original plant to produce the essence and flavor.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> scientific research institute of forestry in Guizhou province

<120> fungus for producing aroma in radix cynanchi wilfordii and application thereof

<130> 2020

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 1

tccgtaggtg aacctgcgg 19

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 2

tcctccgctt attgatatgc 20

<210> 3

<211> 866

<212> DNA

<213> Pandanthus microphylla root endophytic fungi (Tulasnella. sp XYDL003)

<400> 3

ggtgttcgat catgacgttc tattccgtcg tcctcgggac gttaaggcgc tctggtcgag 60

gataaacgac ccctctgacc gaggttaagc ggtcgctgcc tgtgttacct cttccgaggc 120

acacgttaaa gatcgttccg cgttgtgagt ctaacaccag ttgtaacttt ttacaaccgg 180

cagcgctgga tcccttggca cgtcattcga tgaagaccgt tgcaaattgc gataaagtga 240

tgtgatgcgc aagtccacca cttatacgtg aatcatcgag ttgttgaacg cactgcaccg 300

cgccctaatc cggctgcggt atgccccttt gagcgtcatt gtattccttc gggagtcttt 360

tccttgcgaa agacccgagt tcggagtcct cggtctttgg atcgtgttct ctcagatgcg 420

tcgcgccgat cgcctgatgg gtactccaat gcctgagcgt ggagtccctc ggagtttgag 480

acgcgcttga ccggccgttg ggctcgcgtc accaagtccg cgcccttaag gaactccgga 540

ctaccaccca tggactccat ggggtaagac aacccggcag aactaagcat aaccaaaacc 600

gggaggaaat aatctttatc ccccttcacc ggccaatccg ggcgccccgg ggccatgaag 660

gaaacggccc caggaataac gagcccagtg gacccaggtt gaggcgtccc cgctgaggat 720

aacttataca aaggacccgt aaaggatgta cgaattgatt attcattgta cctgtcagcc 780

gactcgctga tcttgcacta tcagaaaacc ggtgcatgca tactgatgga tgcatccagt 840

aactgacttg caagtggtga gcatgc 866

<210> 4

<211> 620

<212> DNA

<213> Pandanthus microphylla root endophytic fungi (Tulasnella. sp XYDL040)

<400> 4

gggtaatcgt catgacgttc tattccgtcg tcctcgggac gttaaggcgc tctggtcgag 60

gataaacgac ccctctgacc gaggttaagc ggtcgccgcc tgtgttacct cttccgaggc 120

acacgttaaa gatcgttccg cgttgtgagt ctaacaccag ttgtaacttt ttacaaccgg 180

cagcgctgga tcccttggca cgtcattcga tgaagaccgt tgcaaattgc gataaagtga 240

tgtgatgcgc aagtccacca cttatacgtg aatcatcgag ttgttgaacg cactgcaccg 300

cgccctaatc cggctgcggt atgccccttt gagcgtcatt gtattccttc gggagtcttt 360

tccttgcgaa agacccgagt tcggagtcct cggtctttgg atcgtgttct ctcagatgcg 420

tcgcgccgat cgcctgatgg gtactccaat gcctgagcgt ggagtccctc ggagtttgag 480

acgcgcttga ccggccgttg ggctcgcgtc gccaagtccg cgtcctttgg gacgtcggta 540

ctacaacgca tgacctcatt ggggtaggac aacccgctag acttaagcat atcaataaag 600

cgggaaggaa atcccagatc 620

Claims

1. A group of fungus producing aroma in the root of the Pandanum parvifolium is characterized in that the two strains are named Tulasnella. sp XYDL003 and Tulasnella. sp XYDL040, the two strains are preserved in the China center for type culture Collection in 5-15 th of 2020, the preservation numbers are CCTCC M2020122 and CCTCC M2020123 respectively, and the fungus producing aroma in the root of the Pandanum parvifolium is Tulasnella. sp XYDL040 is characterized in that: the partition is a barrel hole diaphragm, and the barrel hole covering pad is not provided with a through hole and is arc-shaped; on a PDA culture medium, the colony is milky white at the early stage, grows by adhering to the culture medium, and aerial hyphae are undeveloped; the later stage is light brown, the edge is velvet-shaped, irregular and jagged, and aerial hyphae are developed and protrude; the growth speed of hyphae is 0.10-0.13mm/h, the hyphae branches at right angles or nearly at right angles, the hyphae and the candida cells are accumulated into a pile, the candida cells are circular or elliptical, the size is 6.79-16.39 multiplied by 11.63-45.68 microns, the chain length of the candida cells is generally 4-5 cells, the hyphae have the diameter of 4.39-10.52 microns and the cell length is 61.62-126.21 microns, and the distance between the first partition of the branch and the branch point is 2.23-9.15 microns;

2. The fungus for producing fragrance in the root of paphiopedilum parvifolium as claimed in claim 1, wherein the fungus is isolated from the root system of paphiopedilum parvifolium.

3. Use of the fungus producing aroma in the root of blattbulume as claimed in claim 1 in preparing flavors and fragrances.

4. Use according to claim 3, characterized in that the volatile substance of the aroma-producing fungus Tulasnella sp XYDL040 in the roots of Bidens parviflora consists of: gamma decalactone 78.57%; 2.90 percent of ethanol; 0.32 percent of 2, 3-butanedione; 0.23 percent of 3-methyl-3-butene-1-ol; 0.30% of hexanal; 3.05% of trans-alpha-bergamotene; 0.73% of beta-bisabolene; other undetermined 8.83%;

5. The use according to claim 3, wherein the main volatile substance of the fungus Tulasnella. sp XYDL003 and Tulasnella. sp XYDL040 which produce aroma in the roots of the Bidens parviflora is propiolactone in an amount of 96.79% and 78.57%, respectively.