Method for identifying endophytic fungi at different parts of citral type cinnamomum bodinieri and analyzing metabolic difference

Abstract

The invention discloses a citral type cinnamomum bodinieri different part endophytic fungus identification and metabolic difference analysis method, which comprises the following steps: collecting citral type cinnamomum bodinieri stem and leaf samples, carrying out surface disinfection, separating and purifying endophytic fungi, identifying strains through morphological observation and ITS sequence analysis, carrying out fermentation culture, extracting metabolites by using ethyl acetate, and separating and purifying the metabolites to obtain the citral type cinnamomum bodinieri different part endophytic fungi. And analyzing the difference by using GC-MS. According to the method, 23 endophytic fungi belonging to 9 genus are separated from stems and leaves of cinnamomum bodinieri, 4 citral-producing strains such as CJ5 and CY5 are screened from 8 genus of stems and 9 genus of leaves, the relative content of citral in CJ5 reaches 7.76%, and CY5 contains citral and linalool at the same time. The method disclosed by the invention reveals diversity and metabolic difference of endophytic fungi of cinnamomum bodinieri, and provides an effective means for functional strain screening and resource development.

Description

Technical Field

[0001] The present invention relates to the technical field of plant endophytic fungi separation and identification and metabolite analysis, and particularly to a method for identifying endophytic fungi in different parts of citral-type Cinnamomum camphora and analyzing metabolic differences. Background Art

[0002] Plant endophytes are fungi that live within healthy plant tissues during certain or all stages of their life cycle without causing significant disease in the host plant. The symbiotic relationship between endophytes and their host plants enables them to produce a variety of biologically active secondary metabolites, which have important applications in medicine, agriculture, and industry.

[0003] As an important economic and ecological tree species, the study of endophytic fungi in Cinnamomum camphora has attracted extensive attention. Camphora bodinieri Research on endophytic fungi remains relatively scarce, particularly regarding the diversity and metabolic differences of endophytic fungi in different plant parts (stems and leaves). Existing methods for isolating and identifying endophytic fungi in Cinnamomum plants suffer from low efficiency and accuracy, and lack specificity in analyzing their secondary metabolites, making it difficult to effectively screen for strains with specific functions (such as citral production).

[0004] In addition, citral, as an important terpenoid compound, possesses multiple biological activities such as antibacterial, anti-inflammatory, and antioxidant activities, and has broad application prospects in the fields of food, cosmetics, and medicine. However, the current sources of natural citral are limited, and screening for citral-producing strains from plant endophytes has become an important approach to addressing resource shortages. Therefore, establishing a systematic method for identifying citral-producing endophytic fungi from different parts of Monkey Cinnamomum camphora and analyzing their metabolic differences is of great practical significance for exploring Monkey Cinnamomum camphora endophytic fungal resources and screening for functional strains. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for identifying endophytic fungi in different parts of citral-type monkey camphor and analyzing metabolic differences, so as to solve the existing problems.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: The method for identifying endophytic fungi in different parts of citral-type monkey camphor and analyzing metabolic differences includes the following steps: step 1: sample collection, collecting healthy stem and leaf tissues of citral-type monkey camphor as samples; step 2: surface disinfection, performing surface disinfection on the collected stem and leaf samples; step 3: separation and purification of endophytic fungi, using tissue block separation method to separate endophytic fungi from the disinfected stem and leaf tissues, and performing purification culture; step 4: strain identification, classifying and identifying the purified endophytic fungi through morphological observation and ITS sequence analysis; step 5: fermentation culture, fermenting and culturing the identified endophytic fungal strains; step 6: metabolite extraction, using ethyl acetate to extract secondary metabolites in the fermentation broth; step 7: metabolite analysis, using gas chromatography-mass spectrometry (GC-MS) to analyze the extracts and compare the differences in endophytic fungal metabolites in the stem and leaf parts.

[0007] Further solution: The surface disinfection treatment in step 2 is specifically as follows: first soak in 75% ethanol for 1 minute, rinse with sterile water 3 times, then soak the stems in 7.5% sodium hypochlorite for 20 minutes, soak the leaves in 5% sodium hypochlorite for 20 minutes, and finally rinse with sterile water 3 times.

[0008] Further solution: The tissue block separation method in step 3 is specifically as follows: the disinfected stem and leaf tissues are cut into small pieces of 1 cm×1 cm, inoculated into PDA culture medium, cultured in a constant temperature incubator at 26°C for 7 days, and single colonies are picked for purification.

[0009] Further solution: The ITS sequence analysis in step 4 is specifically as follows: extracting endophytic fungal DNA, performing PCR amplification using universal primers, sequencing the PCR products, and performing Blast comparison analysis with the GenBank database.

[0010] Further solution: The fermentation culture conditions in step 5 are as follows: the endophytic fungus is inoculated into PDB culture medium and cultured at room temperature with shaking at 150 rpm for 7 days. Further solution: The GC-MS analysis conditions in step 7 are as follows: a DB-WAXUI column (5 m × 250 μm × 0.25 μm), helium as the carrier gas, an initial column temperature of 80°C, programmed to 250°C, an electron impact ion source, and a scan range of 50-300 m / z.

[0011] A further solution also includes the step of screening citral-producing endophytic fungal strains: screening out endophytic fungal strains containing citral in the fermentation broth through GC-MS analysis results.

[0012] Further solution: the citral-producing endophytic fungal strain includes Botryosphaeria dothidea strains CJ5 and CY5 and Fungalsp. strains CJ17 and CY21.

[0013] Further plans also include steps to compare the diversity of endophytic fungi in stems and leaves: counting the number of endophytic fungal genera and unique genera isolated from stems and leaves, and analyzing the distribution differences of endophytic fungi in different parts.

[0014] Further scheme: also includes a metabolite functional analysis step: functional classification of the metabolites identified by GC-MS, and analysis of their correlation with the host monkey camphor essential oil components.

[0015] The present invention has the following beneficial effects: This study, the first systematic study of endophytic fungi from different parts (stems and leaves) of the citral-type Cinnamomum camphora, isolated and purified 23 strains belonging to nine genera, revealing the rich diversity of Cinnamomum camphora endophytes. Eight genera were isolated from the stems, two of which were unique to these parts; nine genera were isolated from the leaves, three of which were unique to these parts. This study provides foundational data for the establishment of a Cinnamomum camphora endophyte resource library.

[0016] 2. The present invention adopts a method combining morphological observation with ITS sequence analysis to improve the accuracy and reliability of endophytic fungi identification, laying a foundation for the subsequent screening of functional strains.

[0017] 3. In the present invention, the secondary metabolites of endophytic fungi were analyzed by GC-MS technology, and four citral-producing strains were successfully screened out. Among them, the relative citral content of strain CJ5 was as high as 7.76%, and strain CY5 contained both citral (2.28%) and linalool (8.7%), providing a potential bacterial resource for the biosynthesis of natural citral.

[0018] 4. The present invention can effectively analyze the metabolic differences of endophytic fungi in different parts of Monkey Cinnamomum camphora. It was found that in addition to citral, the metabolites of endophytic fungi also contain serinol, diketopiperazines, nitrogen-containing heterocyclic compounds and other active compounds, providing clues for the discovery of new bioactive substances.

[0019] 5. The method established in the present invention is simple to operate and has good reproducibility. It can be widely applied to the study of endophytic fungi in other Cinnamomum plants, providing technical support for the development and utilization of plant endophytic fungi resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a heat map of the relationship between the isolation sites and fungal species in the present invention.

[0021] Figure 2 is the number of fungal species from different isolated parts in the present invention.

[0022] Figure 3 This is the GC-MS total ion current diagram of the monkey camphor essential oil in the present invention.

[0023] Figure 4This is the GC-MS total ion current diagram of the fermentation broth of the CJ5 strain in the present invention.

[0024] Figure 5 This is the GC-MS total ion current diagram of the CY5 fermentation broth in the present invention.

[0025] Figure 6 This is the GC-MS total ion current diagram of the CY21 fermentation broth in the present invention.

[0026] Figure 7 This is the GC-MS total ion current diagram of the CJ17 fermentation broth in the present invention. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0028] In this study, 23 endophytic fungi were isolated and purified from healthy stems and leaves of Cinnamomum camphora collected in summer using laboratory culture medium and conventional PDA culture medium. Molecular biological identification results (see Table 1) showed that, 13 endophytic fungi were purified, excluding the same strains. These strains belonged to the genus Colletotrichum ( Colletotrichum ), the genus Psoralea corylifolia ( Purpureocillium ), Scopulariopsis ( Sarocladium ), Xylaria ( Daldinia ), Botrytis cinerea ( Botryosphaeria ), Phyllosticta ( Phyllosticta ), Alternaria ( Alternaria ), the genus Phialospora ( Phoma ) and Fusilis spp. ( Neofusicoccum ) were identified from at least nine different genera. Furthermore, two strains (XC9 and XP8) can only be tentatively classified as fungi or endophytes, and their specific taxonomic status awaits further clarification. Overall, the endophytic fungi in Cinnamomum camphora exhibit a considerable degree of taxonomic diversity.

[0029] Table 1 Identification results of endophytic fungi isolated from the stems and leaves of Cinnamomum camphora

[0030] Endophytic fungi isolated from different parts From Table 1 and Figure 1 - Figure 2 The data showed that there were significant differences in the results of endophytic fungi isolated from stems and leaves; Endophytic fungi in stems: A total of 8 genera were isolated from the stems, including the genus Scopulariopsis ( Sarocladium ), Carbonaceous genus ( Daldinia ), Botrytis cinerea ( Botryosphaeria ), Anthrax spp. ( Colletotrichum ), Alternaria ( Alternaria), Phyllosticta ( Phyllosticta ), the genus Fusilis ( Neofusicoccum ) and unidentified fungi.

[0031] There are two genera unique to the stem: Sarocladium ) and Carbonaria ( Daldinia ), suggesting that the stem environment may provide unique colonization conditions for these fungi.

[0032] Botrytis cinerea ( Botryosphaeria ) appeared more frequently in the stems (XC7 and XP3), indicating that it may have a long-term symbiotic relationship with the stem tissues of Cinnamomum camphora; A total of 9 genera were isolated, including the genus Psoralea corymbosa ( Purpureocillium ), Phyllosticta ( Phyllosticta )、Phyllostachys capitalensis( Phyllosticta capitalensis ), Botrytis cinerea ( Botryosphaeria ), Anthrax spp. ( Colletotrichum ), Alternaria ( Alternaria ), Candida spp. ( Phoma ), unidentified fungi ( Fungal sp.) and endophytic fungi of unidentified genera.

[0033] There are 3 genera unique to the leaves: Purpureocillium )、Phyllostachys capitalensis( Phyllosticta capitalensis ) and endophytic fungi were not identified, indicating that the leaf microenvironment may be more conducive to the growth of these fungi.

[0034] Among them, X. purpurogenum (XC2) was isolated only from leaves, which may be related to its preference for leaf surface or stomata; Table 2 Morphological characteristics of endophytic fungi

[0035]

[0036] Dominant bacterial genera and tissue distribution In terms of identification results, the genus Botrytis ( Botryosphaeria ,in particular B. dothidea ) and Colletotrichum spp. ( Colletotrichum sp.) appeared more frequently in the strains isolated in this study, appearing 3 times each, which indicates that they may be a relatively common endophytic fungal group in Cinnamomum camphora tissues. Alternaria alternata ) and Fusilis spp. ( Neofusicoccum parvum) also appeared twice each. These common genera contain many known plant pathogens. Their asymptomatic endophytic presence in healthy Cinnamomum camphora tissues is a common phenomenon in endophyte research. The relationships between these strains and their hosts, such as mutualism, potential pathogenicity, or latent infection, await further functional experimental studies.

[0037] In terms of tissue distribution, this study isolated endophytic fungi from both stems and leaves. Some strains of the genera, such as Botryosphaeria, Colletotrichum, Alternaria, and Fusarium, were found in both stems and leaves, or were isolated from mixed stem and leaf samples (e.g., XP5, XP6, and XP9). This suggests that these fungi possess systemic colonization or widespread distribution within Cinnamomum camphora plants, rather than strict tissue specificity. However, the isolation of Xanthoceras lilacinus (XC2) exclusively from leaves and Colletotrichum lilacinus (XC6) exclusively from stems suggests that some fungi exhibit tissue preferences. This requires further confirmation with larger sample sizes of endophytic fungi from stems, leaves, and even roots.

[0038] In summary, the isolation and purification of endophytic fungi in Cinnamomum camphora preliminarily revealed the diversity of endophytic fungi in the stems and leaves of Cinnamomum camphora in summer, and identified at least 9 fungal genera. Botryosphaeria and Colletotrichum These are likely to be relatively common endophytic fungal groups in this host. The results also demonstrate the importance of culture medium selection for endophytic fungal isolation. For some strains identified only to the genus level or whose taxonomic status remains unclear, such as XC9 and XP8, further investigation combining subtle morphological observations with molecular biological techniques (such as ITS sequence analysis) is needed for more accurate identification. Future research could explore the variations in the endophytic fungal community structure of Cinnamomum camphora under different seasons and locations, and further explore the secondary metabolites of these Cinnamomum camphora endophytes and their interactions with the host.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for identifying endophytic fungi in different parts of Citral-type Cinnamomum camphora and analyzing metabolic differences, characterized in that: The following steps are involved: Step 1, sample collection: collect healthy stem and leaf tissues of Citral-type Cinnamomum camphora as samples; Step 2, surface disinfection: surface disinfection of the collected stem and leaf samples; Step 3, isolation and purification of endophytic fungi: using tissue block separation method to isolate endophytic fungi from the disinfected stem and leaf tissues, and purify and culture them; Step 4, strain identification: Classification and identification of the purified endophytic fungi by morphological observation and ITS sequence analysis; Step 5, fermentation culture: fermenting and culturing the identified endophytic fungal strain; Step 6, metabolite extraction: extracting secondary metabolites in the fermentation broth with ethyl acetate; Step 7, metabolite analysis: The extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) to compare the differences in the metabolites of endophytic fungi in the stems and leaves.

2. The method for identifying endophytic fungi in different parts of Citral-type Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that, The surface disinfection treatment in step 2 is specifically as follows: first soak in 75% ethanol for 1 minute, rinse with sterile water 3 times, then soak the stems in 7.5% sodium hypochlorite for 20 minutes, soak the leaves in 5% sodium hypochlorite for 20 minutes, and finally rinse with sterile water 3 times.

3. The method for identifying endophytic fungi in different parts of citral-type citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that, The tissue block separation method in step 3 is specifically as follows: the disinfected stem and leaf tissues are cut into small segments / pieces of 1 cm×1 cm, inoculated into PDA culture medium, cultured in a constant temperature incubator at 26° C. for 7 days, and single colonies are picked for purification.

4. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The ITS sequence analysis in step 4 specifically includes: extracting endophytic fungal DNA, performing PCR amplification using universal primers, sequencing the PCR products, and performing Blast comparison analysis with the GenBank database.

5. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The fermentation culture conditions in step 5 are as follows: the endophytic fungus is inoculated into the PDB culture medium and cultured at room temperature with shaking at 150 rpm for 7 days.

6. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The GC-MS analysis conditions in step seven are: DB-WAXUI chromatographic column, 5m×250μm×0.25μm, helium carrier gas, column box initial temperature 80°C, programmed temperature increase to 250°C, electron bombardment ion source, scanning range 50-300m / z.

7. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The method also includes the step of screening citral-producing endophytic fungal strains: screening out endophytic fungal strains containing citral in the fermentation liquid through GC-MS analysis results.

8. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The citral-producing endophytic fungal strain comprises Botryosphaeria dothidea strains CJ5, CY5 and Fungal sp. strains CJ17, CY21.

9. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: It also includes steps to compare the diversity of endophytic fungi in stem and leaf parts: counting the number of endophytic fungal genera and unique genera isolated from stem and leaf parts, and analyzing the distribution differences of endophytic fungi in different parts.

10. The method for identifying endophytic fungi in different parts of Citral-type Monkey Cinnamomum camphora and analyzing metabolic differences according to claim 1, characterized in that: The method also includes a metabolite functional analysis step: functional classification of the metabolites identified by GC-MS and analysis of their correlation with the components of the host monkey camphor essential oil.

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