CN109370947B - Enterobacteriaceae and its application in preventing and controlling Aspergillus flavus and toxins in peanut during storage period - Google Patents

Abstract

The invention discloses an Enterobacter sp. Vt-7, the deposit number of which is CCTCC M 2018719. Its volatile metabolites 1-pentanol and phenethyl alcohol. At the same time, the invention also discloses the application of the Enterobacter Vt-7 and its volatile metabolites to the prevention and control of Aspergillus flavus and toxins during storage period, which can effectively inhibit the occurrence of Aspergillus flavus disease in peanuts under airtight environments with different water activities. Contaminated with aflatoxins. At the same time, the present invention also reports the broad-spectrum bacteriostatic effect of the volatile substances produced by Vt-7, which can inhibit the growth of eight important fungal diseases.

Description

Enterobacter and application thereof in preventing and treating peanut aspergillus flavus and toxin in storage period

Technical Field

The invention belongs to the technical field of biological control of plant diseases, and particularly relates to enterobacter and application thereof in control of peanut aspergillus flavus and toxin in a storage period.

Background

Aspergillus flavus is a common fungal disease in nature, can be harmful in fields and storage periods, and infects various crops such as peanuts, corns, soybean rice and the like, so that serious economic loss is caused. In addition, in the infection process, the aspergillus flavus can also generate high-toxicity aflatoxin, which seriously harms human health. Aflatoxins have been classified as a carcinogen by the World Health Organization (WHO) since 1993. The aflatoxin has a stable structure and strong acid, alkali and heat resistance, and cannot be completely removed after being sterilized at high temperature for 20 hours. So far, more than 20 aflatoxins have been found, the aflatoxin B1(AFB1) has the highest toxicity, which is 10 times that of potassium cyanide and 68 times that of arsenic. AFB1 is also a strong carcinogen, is easy to induce serious human pathological changes such as liver cancer, kidney cancer and rectal cancer, and is the strongest carcinogen identified by the World Health Organization (WHO). About 37 thousands of people die of liver cancer in China every year, accounting for more than 50% of the death rate of liver cancer in the world, and the causes of the diseases are directly related to the pollution of AFT. In 2004, severe AFT poisoning occurred in the eastern part of kenyas, causing 317 liver failure lesions and 125 deaths, the cause of death was directly related to food crops that had been contaminated with aflatoxin during storage. In recent years, with the increasing concern of people on the harm of aflatoxin in China, the exceeding-standard event of aflatoxin is exposed successively. In 2011, 24 months and 12 months, aflatoxin M1 of a batch of products produced by Mongolian dairy industry (Meishan) limited company exceeds 140 percent. In vegetable oil products of 12 months and 27 days in 2011, part of 3 products in Guangdong province cannot be checked in batches, and the reasons are that aflatoxin B1 indexes are unqualified.

In view of the tremendous threat of aflatoxins to humans and animals, WHO stipulates: the maximum allowable concentration of aflatoxin in the food product is 15 mug/kg. The related law of the federal government stipulates that the content of aflatoxin (which refers to the total amount of B1+ B2+ G1+ G2) in foods for human consumption and dairy feeds cannot exceed 15 mug/kg, the content of milk for human consumption cannot exceed 0.5 mug/kg, and the content of other animal feeds cannot exceed 300 mug/kg. The national regulation of European Union is stricter, and the content of aflatoxin b1 in consumer goods for human life cannot exceed 0.05 mug/kg. According to the regulations of China, the maximum limit of AFB1 in corn, peanut cake (meal) and rapeseed cake (meal) is 50 mug/kg, and the maximum limit of AFB1 in peanuts directly eaten by human is 20 mug/kg.

Strict food limit standards reduce aflatoxin pollution to a certain extent, but cannot fundamentally control the generation of toxins, and how to reduce the generation and harm of toxins from the source is a hotspot of current research. In recent years, microorganisms are utilized to prevent and control aspergillus flavus and toxins, the development is rapid, so far, a plurality of microorganisms are reported to have high-efficiency biocontrol effect, particularly, the microorganisms are utilized to generate volatile gas substances, the effect is obvious in the application of preventing and controlling the aflatoxin in the storage period, and the microorganisms can generate volatile antibacterial substances, such as Shewanella alga YM8 reported in the early period, and the harm and toxin pollution of the aspergillus flavus and corn in the storage period are inhibited. The microbial source volatile gas has the advantages of simple use, uniform and quick dispersion, wide contact surface and the like, and has better application prospect in the prevention and treatment of fungal diseases and toxins in the storage period. However, the storage period control of aflatoxin and toxin is still the starting stage, the screened microbial strains and bacteriostatic metabolic substances are few, and in order to further expand the utilization efficiency of microorganisms and improve the action effect, bacteriostatic microbial resources are screened from the nature, the gas production characteristics and the bacteriostatic action of the bacteriostatic microbial resources are researched, and the storage period aflatoxin control research is developed.

Disclosure of Invention

The invention aims to provide an Enterobacter (Enterobacter sp.) Vt-7 and application thereof. Vt-7 and the volatile metabolite produced by the Vt-7 can effectively inhibit the infection of the peanut aspergillus flavus and the production of toxin in the storage period, and have high-efficiency broad-spectrum bacteriostasis to inhibit the growth of various pathogenic fungi.

Based on the purpose, the invention adopts the following technical scheme:

enterobacter (Enterobacter sp.) Vt-7 with a preservation number of CCTCC M2018719.

The 16S rDNA sequence of this enterobacterium is shown in table SEQ ID NO: 1.

The enterobacter is applied to inhibiting the growth of various pathogenic fungi and inhibiting the aspergillus flavus and toxin of crops in the storage period.

The invention collects soil from a Yunshan tea garden of Xinyang city, Henan province, China, and separates an enterobacter strain with high-efficiency inhibition effect on aspergillus flavus from the soil by a microbiological method. The strain is named as Enterobacter Vt-7, (Enterobacter sp. Vt-7), and is delivered to China Center for Type Culture Collection (CCTCC) of Wuhan, China at 29/10 in 2018, with the preservation number of CCTCC M2018719.

Bacteriological characteristics of Enterobacter (Enterobacter sp.) Vt-7:

enterobacter Vt-7 is a gram-negative bacterium, and can utilize D-glucose, D-fructose, sucrose, L-arabinose, D-mannose, etc., and can utilize citric acid, cellulose, starch and hydrogen sulfide.

The 16S rDNA sequence of the strain Vt-7 is sequenced and analyzed, and the nucleotide sequence is shown as a sequence table SEQ ID NO. 1.

The invention detects the high-efficiency inhibition effect of the strain Vt-7 on aspergillus flavus hyphae and other seven fungi hyphae. Volatile bacteriostatic metabolites generated by Vt-7 are identified, and relevant researches such as the inhibition effect of Vt-7 on the aspergillus flavus and toxin residue analysis are analyzed under the condition of closed storage.

The enterobacter Vt-7 disclosed by the invention has a good bacteriostatic action, and can inhibit the morbidity and toxin production of the peanut aspergillus flavus under a closed storage environment condition. The strain can be applied to control the mould pollution of peanuts in the storage period, and can be extended to be applied to safe storage and transportation of other grain crops, fruits and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) the enterobacter Vt-7 separated by the invention is soil bacteria, and the high-efficiency antibacterial microorganism separated from the soil can be used as a biocontrol strain for fungal diseases in the storage period of crops;

(2) the invention proves that the separated enterobacter Vt-7 can produce volatile antibacterial substances for the first time, and can effectively inhibit the growth of aspergillus flavus hyphae in a closed environment;

(3) the separated enterobacter Vt-7 has a remarkable bacteriostatic effect, and can efficiently inhibit the incidence of aspergillus flavus on peanuts and the synthesis of toxins in a closed space;

(4) the enterobacter Vt-7 obtained by the invention can generate volatile antibacterial substances, and the key antibacterial substances of 1-pentanol and phenethyl alcohol are determined by a gas chromatography-mass spectrometer;

(5) the 1-pentanol and phenethyl alcohol generated by the enterobacter Vt-7 obtained by the invention have obvious bacteriostatic effect, can completely inhibit the growth of aspergillus flavus hyphae and spore germination under the concentration of 200 mu g/L (material weight/space volume), and has obvious bacteriostatic effect.

Description of the drawings:

vt-7 preservation date: 29/10/2018, depository: china Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M2018719.

Sequence listing SEQ ID NO:1 is the 16S rDNA sequence of Enterobacter Vt-7 isolated in accordance with the present invention;

FIG. 1 is a phylogenetic tree analysis of the 16s rDNA sequences of Enterobacter Vt-7 strain and its allied species;

FIG. 2 is a graph showing the inhibitory effect of Enterobacter Vt-7 on the growth of AF hyphae;

FIG. 3 is a graph of Aspergillus flavus control of peanut kernels with different water activities by Enterobacter Vt-7; in FIG. 3, CK represents the control group of Aspergillus flavus, Vt-7+ CK represents the control group treated by adding Vt-7 strain, and the disease onset state is shown after culturing at 28 ℃ for 5 days in a closed state;

FIG. 4 is an analysis of the inhibition of Enterobacter Vt-7 by the addition of activated carbon; the upper bar graph is the hyphal diameter counted after 5 days of culture; in the lower graph, CK is an inoculated aspergillus flavus control group, CK + C aspergillus flavus is added with active carbon for treatment, CK + Vt-7 is added with Vt-7 for aspergillus flavus, and CK + C + Vt-7 is subjected to bacteriostatic effect analysis in the presence of the aspergillus flavus, the aspergillus flavus and the active carbon simultaneously;

FIG. 5 is a scanning electron microscope analysis of Aspergillus flavus on the surface of peanut, the water activity of the peanut kernel is 0.9, Aspergillus flavus spores (CK) are inoculated, and the inoculated peanut kernel and Vt-7 are co-cultured (Vt-7+ CK), and the microscopic structure of the Aspergillus flavus is observed after 5 days. Wherein co represents conidium, cp represents conidium head, and my represents hypha;

FIG. 6 shows the inhibitory effect of Enterobacter Vt-7 on the growth of hyphae of 8 different fungi;

FIG. 7 shows the results of GC-MS detection of volatile substances produced by Enterobacter Vt-7;

FIG. 8 is a minimum inhibitory concentration analysis of Vt-7 volatiles on AF hyphal growth and spore germination; the inhibitory activity of each compound on aspergillus flavus hypha and spores was determined at different dilution concentrations, and the culture time was 4 days.

Detailed Description

The present invention is further illustrated by the following specific examples.

EXAMPLE 1 isolation and bacteriological characterization of Strain Vt-7

(1) Screening of Strain Vt-7

The invention collects soil from a Yunshan tea garden of Xinyang city, Henan province, and separates an enterobacter strain which can produce volatile substances and has high-efficiency inhibition effect on aspergillus flavus by a microbiological method. The candidate strain was numbered Vt-7.

Separating microorganism by NA purification culture method, weighing 1g soil sample, placing in 2mL centrifuge tube, adding 1mL sterile water, mixing, and gradient diluting to 10^-6. And (3) coating 100 mu L of bacterial suspension on the surface of an NA culture medium, culturing for 2-3 days at 28 ℃, selecting single colonies with obvious morphological difference, analyzing the bacteriostatic effect of the single colonies after purification culture, and selecting strains with better bacteriostatic effect for storage for subsequent research.

The NA culture medium comprises the following formula: 3.0g of beef extract, 10.0g of peptone, 5.0g of NaCl and 15.0g of agar, adding distilled water to 1L, and adjusting the pH to 7.2.

(2) Morphological characterization of Vt-7 Strain

After the Vt-7 strain is cultured in the NA culture medium for 48 hours, the colony is viscous, yellow, smooth in surface, opaque and gram-negative.

(3) Molecular biological characterization of Vt-7

Selecting a Vt-7 single colony, inoculating the single colony to 20mL of NB culture solution (3.0 g of beef extract, 10.0g of peptone and 5.0g of NaCl, supplementing distilled water to 1L, adjusting the pH to 7.2), shaking for 24h, centrifuging at 12000rpm to collect thalli, extracting genomic DNA by adopting 10mM Tris-HCl (purchased from Amresco) and 1mM EDTA, extracting by using phenol-chloroform-isoamylol (25:24:1) after extraction, adding 3mol/L NaAc and 2 times volume of absolute ethyl alcohol, shaking for standing, washing the precipitate by using 70% ethyl alcohol, drying in the air, and dissolving by using TER. The conserved sequence was amplified using 16S rDNA specific primers and sent to Wuhan Tianyihui biology Inc. for sequencing. The length of the sequencing sequence is 1420bp, and the specific sequence is shown as SEQ ID No.: 1 is shown.

The sequencing primer is as follows: 27F: AGAGTTTGATCCTGGCTC

1541R：AAGGAGGTGATCCAGCCGCA

In Genbank database, 16s rDNA sequence of Vt-7 strain was subjected to BLAST search, and as a result, it was revealed that Vt-7 has high homology with Enterobacter, which was preliminarily determined to be Enterobacter bacteria, and further, strains having homology higher than 97% with Vt-7 were selected from the genus, and phylogenetic tree analysis was performed.

According to the phylogenetic analysis of the 16S rDNA gene sequence (figure 1), the Vt-7 cannot be gathered into one branch with the selected high-homology strain and independently forms a single branch, which indicates that the strain Vt-7 is far away from the affinity of the screened strain and is possibly an independent taxon, and the strain is temporarily named as Enterobacter Vt-7 and Enterobacter sp.Vt-7. And the strain is delivered to China Wuhan university China typical microbiological culture collection center (CCTCC) for preservation in 2018, 10 months and 29 days, wherein the preservation number is CCTCC M2018719.

(4) Physiological and biochemical identification of strain WY6-5

To further determine the taxonomic status of the strain, BIOLOG Microstation was used^TMAnd the System microorganism identification instrument analyzes physiological and biochemical characteristics. Activating the strain on an NA culture medium, selecting a single colony after 12 hours, inoculating the single colony to IF-A GEN III encapsulating fluid culture solution, uniformly mixing, inoculating the single colony to a Biolog Gen III culture plate, culturing for 10-12 hours at 37 ℃, carrying out physiological and biochemical analysis by a BIOLOG microorganism identification system, carrying out systematic comparison analysis with the identified microorganism, and selecting the species with the highest physiological and biochemical reaction similarity for comparison. The results of the physiological and biochemical tests are shown in Table 1.

The results show that the strain Vt-7 has a recent relationship with the BIOLOG system and is E.aerogenes, compared with the strain Vt-7, the physiological and biochemical reactions of the strain Vt-7 are remarkably different, particularly the strain Vt-7 is negative and the E.aerogenes strain is positive in the utilization of Gelatin, alpha-D-Lactose, D-Arabitol, D-Fucose, L-Fucose, D-Malic Acid, D-Turanose and the like, and the strain Vt-7 and the E.aerogenes have different reaction results in the tolerance experiments of Minocycline, Nalidixic Acid, D-Serine and the like, so that the strain Vt-7 is inferred to have a specific physiological and biochemical reaction and can be a new classification unit.

In summary, through phenotypic observation, molecular biological identification and physiological and biochemical detection and analysis, the strain Vt-7 has obvious differences compared with the existing identified strains, the classification status of the strain cannot be determined, and the strain is probably a new microorganism species, so the strain is named Enterobacter (Enterobacter sp.) temporarily, and the classification status of the strain is further analyzed in later research.

Example 2 bacteriostatic action of Enterobacter Vt-7 on Aspergillus flavus

Experiment for inhibiting growth of aspergillus flavus hyphae by enterobacter Vt-7

The procedure was carried out in a petri dish (diameter 9cm) snap-in manner. Inoculating Aspergillus flavus spores in 50mL of PDB culture solution (peeled potato 200.0g, boiling in boiling water for 20min, filtering with gauze, collecting filtrate, adding glucose 20.0g, diluting with distilled water to constant volume of 1L, adjusting pH), shaking at 28 deg.C and 200rpm for 3 days, selecting white Aspergillus flavus clusters, and inoculating in the center of a culture dish containing PDA. Strain Vt-7 (100. mu.L, OD)₆₀₀1.7) on the surface of NA medium. And buckling the culture dish inoculated with the aspergillus flavus silk blocks on the culture dish coated with Vt-7, and sealing and storing by using transparent adhesive tapes. And (3) taking the treatment of inoculating only the aspergillus flavus mycelia as a control, repeating each treatment for 3 times, culturing for 5 days at 28 ℃ in the dark, calculating the diameters of the mycelia subjected to different treatments and calculating the bacteriostasis rate. The calculation formula of the bacteriostatic rate is as follows:

the bacteriostatic ratio (%) (control hypha diameter-treated hypha diameter)/control hypha diameter × 100.

Analysis of Experimental results

As shown in FIG. 2, it can be seen from FIG. 2 that after 12 hours of cultivation, the hyphae of the control group grew vigorously to cover the surface of the PDA medium, while the Aspergillus flavus hyphae inoculated by the Vt-7 treatment group did not grow, i.e., Vt-7 had a significant bacteriostatic effect on the growth of Aspergillus flavus hyphae, and the inhibition rate reached 100%. In the non-contact culture process of the enterobacter Vt-7 and the aspergillus flavus hyphae in the sealed culture dish, volatile gaseous substances can be generated, and the growth of the aspergillus flavus hyphae is completely inhibited.

Example 3 Enterobacter Vt-7 activated carbon adsorption experiment

The method comprises the following steps: and (4) performing buckling culture by adopting a separation dish (the culture dish cover adopts a non-separation dish and is used for inoculating fungi, and the culture dish bottom adopts a separation dish and is used for coating bacteria and adding activated carbon). The activated Carbon (Carbon, C) experiments were divided into four groups for treatment, namely CK, CK + C, CK + Vt-7, CK + C + Vt-7. Pouring 15mL of PDA culture medium into the four groups of treated non-separated dish covers, inoculating Aspergillus flavus mycelium pellets, and adding active carbon or Vt-7 bacteria liquid into the bottoms of the separated dishes respectively. 6.0g of active carbon is put into one side of the bottom of the CK + C separation dish, and the other side of the CK + C separation dish is blank; the bottom of the CK + Vt-7 separation dish is poured with NA culture medium, uniformly coated with Vt-7(50 mu L, OD600 is 1.7), and the other side is blank; the bottom of the CK + C + Vt-7 dish was filled with NA medium and uniformly coated with Vt-7(50 μ L, OD600 ═ 1.7), and 6.0g of activated charcoal was added to the other side. PDA medium inoculated with AF mycelial blocks was cultured in a cross-hatch manner in all treatments. Sealing with adhesive tape, storing, and culturing in 30 deg.C incubator. There were 2 replicates per treatment. And after 5 days of culture, counting the diameter of the hyphae and calculating the bacteriostasis rate.

The results of the activated carbon adsorption experiments are shown in fig. 3, the mycelia of aspergillus flavus in the four treatments all grow, particularly in CK and CK + C treatment groups, the mycelia grow fastest, the diameter of the mycelia reaches 5.2cm after 5 days of culture, and no significant difference exists between the mycelia and the CK + C treatment groups, which indicates that the activated carbon has no inhibition effect on the growth of aspergillus flavus; when the treatment of Vt-7 is added, the growth of aspergillus flavus hyphae is obviously inhibited, and in the treatment of CK + Vt-7, the diameter of the hyphae is only 0.8cm, which shows that volatile gas generated by Vt-7 can inhibit the extension of the aspergillus flavus hyphae; the diameter of the hyphae in the CK + C + Vt-7 treated group is larger than that of CK + Vt-7, but smaller than that in the CK and CK + C treated groups, which shows that the activated carbon has adsorption effect, and can absorb volatile gas generated by Vt-7, so that the concentration of volatile substances in the whole culture space is reduced, and the inhibition effect on the growth of AF hypha blocks is obviously reduced. The active carbon adsorption experiment fully proves that the volatile gas substances generated by the enterobacter Vt-7 are the root cause for completely inhibiting the growth of aspergillus flavus hyphae.

Example 4 prevention and treatment Effect of Enterobacter Vt-7 on Aspergillus flavus and toxin of peanut

Sample preparation:

1) weighing 100g of peanut seeds in 2 triangular bottles of 250mL respectively, sterilizing at 121 ℃ and 1.01MPa for 20min, standing at room temperature, and cooling; 2) all flasks were inoculated with freshly collected A.flavusSeed liquid 1mL (5X 10)⁵cfu/mL), shaking for 10 min; 3) proper amount of sterilized water is added into the two triangular bottles respectively, and the water activity is adjusted to be 0.8 and 0.9.

The treatment method comprises the following steps:

peanut inoculation experiments were divided into two water activity treatments, 0.8 and 0.9, each water activity treatment was divided into Control (CK) and Vt-7 groups, and all experiments were performed using a desiccator. 40mL of NA medium was poured into the bottom of the desiccator, and after coagulation, Vt-7 (200. mu.L, OD) was added to the surface of the experimental group medium₆₀₀1.7) strain, and uniformly coating on the surface of an NA culture medium, taking a blank NA culture medium as a control, respectively placing the middle parts of an experimental group and the control group into 3 culture dishes, and adding the same weight of peanut seeds inoculated with aspergillus flavus spore liquid into each dish (respectively under the conditions of water activity of 0.8 and 0.9). Sealing vaseline, culturing at 28 deg.C for 5 days, and detecting the incidence of peanut grains in the treated group and the control group under two water activities. And grinding after drying to detect the content of aflatoxin.

Extracting aflatoxin: the ground sample was weighed 1g into a 5mL polypropylene plastic tube. It was extracted with 5mL of hexane/water (84/16, v/v). Screwing the pipe cover, performing vortex treatment for 1min, and performing ultrasonic treatment for 60 min. Centrifuging at 6000rpm for 10min, taking 1mL of supernatant, transferring to a new centrifuge tube, adding n-hexane with the same volume, uniformly mixing, and standing for layering. The lower organic phase was aspirated at 500. mu.L for toxin quantification.

Experimental results and analysis:

after the peanut seeds are inoculated with aspergillus flavus spores and cultured for 5 days (figure 4), the results show that the incidence of aspergillus flavus of the peanut seeds in the control group is obvious, the incidence rate of the peanut seeds is up to 100 percent under two water activities, the green mildew layer is fully distributed on the surface of the peanut seeds, the number of the spores is obviously higher than 0.8 under the culture condition of the water activity of 0.9, and the higher the water activity is, the more beneficial to the incidence of the aspergillus flavus is. Compared with the prior art, the infection and the morbidity of the peanut aspergillus flavus can be effectively inhibited by the treatment of Vt-7, no obvious infection state is seen on the surface of the peanut, and no green spores appear, so that the condition that volatile substances are generated by Vt-7 under a non-contact condition and distributed in upper-layer peanut grains, the germination of the aspergillus flavus spores is effectively inhibited, and the antibacterial effect is obvious.

The detection result of aflatoxin is shown in fig. 3, in the control group of peanuts, both AFB1 and AFB2 are detected, particularly, the concentration of AFB1 is higher, and the content of AFB1 is higher than 600ppb under the condition of water activity of 0.9, and is obviously higher than 0.8; AFB2 was at a lower concentration, 200ppb at 0.9 water activity; indicating that the toxin content increases with increasing water activity. Compared with the Vt-7 treatment groups, the differences of the Vt-7 treatment groups are obvious, and AFB1 and AFB2 are not detected in peanut samples, so that the Vt-7 volatile gas substance has a high-efficiency inhibition effect on toxin synthesis, and the generation of aflatoxin can be effectively inhibited under two water activity conditions.

Example 5 microscopic examination of the inhibition of Aspergillus flavus by Enterobacter Vt-7

In order to detect the structural change of the aspergillus flavus cells, peanut seeds under the condition of high water activity of 0.9 are selected for scanning electron microscope observation. Peanut kernels of the control group and the Vt-7 treated group were taken out, fumigated and fixed in 1% osmate for 1 hour, respectively, a small piece of peanut coat was torn off by tweezers, fixed, then treated with gold spray, and observed by scanning electron microscopy (JSM-6390, Hitachi, Japan). The scanning results are shown in fig. 5.

Scanning electron microscope results show that a large amount of aspergillus flavus mycelia cover the surface of the peanut in the control group, the mycelia generate conidial heads, a large amount of spores are grown on the conidial heads, the spore structures are uniform, and the shapes are full. The Vt-7 treated peanut has only a few spores on the surface, which are original spores for inoculation, no germination of the spores, shriveled surface and uneven structure. The volatile substances generated by Vt-7 can inhibit the germination of aspergillus flavus spores on the surface of peanuts, and further inhibit the spores from germinating to form hypha and conidium heads.

Example 6 broad-spectrum bacteriostasis of Enterobacter Vt-7

The broad-spectrum bacteriostasis experiment is carried out by adopting a culture dish (diameter is 9cm) buckling culture method. And selecting a fungus mycelium block to inoculate in the center of a PDA culture dish. Strain Vt-7 (100. mu.L, OD)₆₀₀1.7) on the surface of another dish containing NA medium. The petri dish inoculated with the fungal hyphae block is buckled on the petri dish coated with Vt-7, the treatment of inoculating only the aspergillus flavus hyphae block is used as a control, and the rubber belt is sealed for storage. Each treatment was repeated 3 times, and after 5 days of culture at 28 ℃ in the dark,and (5) counting the hypha diameter and calculating the bacteriostasis rate.

The formula for calculating the bacteriostasis rate is as follows:

the bacteriostatic ratio (%) (control hypha diameter-treated hypha diameter)/control hypha diameter × 100.

The broad spectrum bacteriostasis test results are shown in FIG. 6, the fungal hyphae in the control group grow normally and rapidly, and no hyphae in the Vt-7 treatment group grow. The Vt-7 strain has obvious bacteriostasis on the growth of pathogenic bacteria, and the bacteriostasis rate is higher than 80%, wherein the bacteriostasis rate on botrytis cinerea, rice blast, soybean anthracnose fusarium graminearum and alternaria alternata is up to 100%, the bacteriostasis rate on aspergillus fumigatus is 97.8%, the bacteriostasis rate on aspergillus flavus is 96.6%, and the bacteriostasis rate on mango anthracnose is 83.1%. Double dish buckling experiments prove that the strain Vt-7 can generate gas secondary metabolites and has good broad-spectrum antibacterial effect.

Example 7 Enterobacter Vt-7 volatile gas detection

The strain Vt-7 is inoculated on the surface of the NA culture medium in a sterile 100mL triangular flask, uniformly coated, sealed and stored, and the triangular flask without Vt-7 is taken as a control. All flasks were incubated in a 37 ℃ incubator for 24h, equilibrated in a 40 ℃ water bath for 10min, and then subjected to sample extraction and GC-MS/MS detection in sequence, with 2 replicates per treatment.

Enrichment of volatile species was performed using a solid phase microextraction column (SPME). Inserting the SPME extraction head into the plastic film, pushing out the fiber head to make the fiber head in the middle position above the sample bottle, and adsorbing for 30 min. And withdrawing the fiber head to the extraction head, pulling out the sample bottle, and transferring to a gas chromatography-mass spectrometer (GC-MS) for sample introduction detection, wherein the detection parameters are designed as follows.

GC-MS/MS conditions:

the temperature of a sample inlet is 250 ℃; the carrier gas is helium, and the column flow rate is 30 mL/min; no split-flow sample introduction. Temperature programming conditions: the initial temperature is 60 deg.C, holding for 2min, heating to 150 deg.C at 5 deg.C/min, holding for 2min, heating to 280 deg.C at 8 deg.C/min, and holding for 2 min. J & WHP-5 MS elastic quartz capillary column (30m × 0.25mm ID, 0.25um thick film).

The ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, and the ionization mode is as follows: EI source with energy of 70eV, and detecting with full scan mode in the detection range of 50-550 amu. Detection of substances the spectral library was automatically retrieved from the National institute of Standards and Technology (NIST 17) and the substances detected were characterized. The detection is shown in fig. 7.

GC-MS/MS detects volatile substances produced by Vt-7, and the volatile substances obtained by deducting the substances in the NA culture medium are specific volatile components produced by Vt-7. As can be seen from FIG. 7, Vt-7 was found to be a total of 3 specific substances in the experiment. Aromatic compounds, alkane compounds and alcohols, all of which are small molecular substances with molecular weight between 88 and 122 daltons (D) and are easy to volatilize. Through analysis, the three substances are taken as main metabolic substances, and the bacteriostatic action of the three substances is analyzed. Only two standards are currently available, so we will perform the next minimum inhibitory concentration test with component 1 (1-pentanol) and component 2 (phenethyl alcohol).

TABLE 2 GC-MS/MS identification of Vt-7 metabolites

Note: and (4) representing the specific volatile substances detected in the Vt-7 group, wherein a is the peak area of the metabolite divided by the sum of the peak areas of all the substances, b/c is the score of the forward comparison and reverse comparison of the spectrogram of the metabolite and the spectrogram in the NIST 17 spectral library, and the highest value is 1000.

Example 81 analysis of the inhibitory Effect of Pentanol and Phenylethanol on Aspergillus flavus

In order to analyze the bacteriostatic action, 1-pentanol and phenethyl alcohol standard substances are purchased for bacteriostatic tests. The inhibition effect of purchased standard products on aspergillus flavus is determined by adopting a double-dish buckling culture method, and experimental objects are divided into AF mycelia and AF conidia.

The specific operation is as follows: and (3) detecting the inhibition effect of the single component on the aspergillus flavus spore germination and hypha growth by adopting a double-dish buckling method. The purchased standard was diluted to 200. mu.g/L, 100. mu.g/L, 10. mu.g/L and 5. mu.g/L (weight of substance/volume of space) in this order, and then subjected to the double reaction with AF mycelium and conidia, respectivelyThe plates were co-cultured in a snap-fit manner with a blank sterile water reagent as a control. Placing a sterile circular filter paper sheet (the diameter is 0.5cm) in a culture dish (the diameter is 9cm) at the lower layer, respectively dropwise adding the single-component diluents with different concentrations on the filter paper sheet, and quickly buckling the PDA culture dish inoculated with the aspergillus flavus silk blocks on the culture dish placed with the filter paper sheet; if inoculated, the spore liquid (5. mu.L, 10)⁵cfu/mL), placing a sterile circular filter paper sheet in the center of a PDA culture dish, inoculating, sealing with adhesive tape, culturing at 28 ℃ for 4 days, measuring the diameter of aspergillus flavus mycelia, and calculating the bacteriostasis rate.

Experimental results and analysis:

after culturing for 4 days, the inhibition effect of the standard substances with different concentrations on the aspergillus flavus is detected, and the specific result is shown in figure 8. FIG. 8 shows that both substances have high bacteriostatic action, and the bacteriostatic action is better as the concentration increases, wherein, when the 1-pentanol is at 200 μ g/L (mass of substance/volume of space), the phenethyl alcohol is at 100 μ g/L, the growth of AF spores can be completely inhibited, which shows that the minimum bacteriostatic concentration for spore germination is respectively 200 μ g/L and 100 μ g/L, the minimum bacteriostatic concentration for hypha growth is slightly higher, the phenethyl alcohol is 200 μ g/L, and the 1-pentanol is higher than 200 μ g/L.

<110> Xinyang college of teachers and schools

<120> enterobacter and application thereof in prevention and treatment of peanut aspergillus flavus and toxin in storage period

GCTACCATGCAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTATTAGGTGGGGTAACGGCTCACCTAGGCGACAATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAAAAGGCCTTCTGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGCTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGCGCCAGCAGCCGCGGTATTACGGAGGGGGTACGCGTTAATCGCAATTACTGGGCATAAACCGCTCGCAGGCGGTCTGTCTTCTCGGATTTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTACCACTTG

Claims (1)

1. Enterobacter sp. Vt-7, its deposit number is CCTCC M 2018719, the nucleic acid sequence of the 16SrDNA of the Enterobacter is shown in SEQ ID NO: 1; the volatile metabolite produced by the Enterobacter is used to inhibit Growth of a variety of pathogenic fungi, and inhibition of Aspergillus flavus and toxins in storage crops.

Images