Pichia kudriavzevii and application of volatile substances of pichia kudriavzevii

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to pichia kudriavzevii and application of volatile substances of the pichia kudriavzevii. According to the invention, an endophyte Pichia delbrueckii Bg7 is separated from strawberry leaves, the endophyte can strongly inhibit the growth of valsa ceratosperma, and the inhibition rate is 58.16%; volatile organic compounds generated by the bacterial strain significantly inhibit the growth of valsa ceratosperma, gray mold of strawberry and physalospora piricola, and the inhibition rates are 64.16%, 100% and 59.92% respectively; volatile organic compounds of the strain Bg7 can completely inhibit occurrence of pear tree rot and strawberry gray mold, the prevention and control effect is remarkable, and the strain Bg7 has important significance in prevention and control of pear tree rot and strawberry gray mold and has potential value in further research and practical application.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Pichia pastoris. Background Art

[0002] Fruit tree planting is an important part of my country's agriculture. The healthy development of the fruit industry is of great significance to promoting the increase of fruit farmers' income and regional economic growth. In the planting industry, the planting area, fruit production and output value of fruit trees rank third after grain and vegetables. However, various fruit tree diseases caused by pathogenic microorganisms such as fungi, bacteria, oomycetes, viruses, and nematodes have led to a decrease in fruit yield and quality, and even caused dead trees, ruined gardens, and no harvests. The economic losses are huge, which seriously affects the healthy and sustainable development of the fruit industry. Taking strawberry, apple and pear as examples, strawberry is a widely planted horticultural crop in my country. During its growth process, it is often infected by various diseases, such as gray mold, powdery mildew, anthracnose, etc., which seriously affect the quality and yield; apple tree rot, ring rot and early leaf fall diseases mainly including brown spot and spotted leaf fall are the three major diseases of apples in my country. They are most common and cause serious damage in my country's apple producing areas; pear tree diseases can occur at all stages of the growth of pear trees, causing leaf shedding, branch death and fruit rot, resulting in tree weakness, which has become a yield reduction factor that cannot be ignored. The diseases that harm pear trees are mainly fungal diseases, such as pear black spot, pear anthracnose, pear black spot and pear rot.

[0003] Chemical control is the most common and most effective method of disease control. However, compared with other crop diseases, fruit tree diseases have a short research history, less investment, more types of diseases, more serious harm, and serious irrational use of pesticides, which brings a series of environmental pollution, pesticide residues and other problems. Eco-friendly biological control methods are becoming more and more popular in the prevention and control of plant diseases. Biological microorganisms can replace chemical agents to reduce harm to human health and the environment, and have advantages such as low residue and low resistance. As a natural control tool, biocontrol strains have broad application prospects. For example, endophytic fungi in plants not only have a certain growth-promoting effect on crops, but also live in plants. This special environment makes them easier to play a role in biocontrol than other saprophytes and epiphytes. Endophytic fungi can also produce a variety of antimicrobial active substances (such as alkalis, peptides, terpenes, phenols, etc.) to inhibit the growth of pathogens. Patent 202310416310.0 discloses a strain of fermented Pichia pastoris LZ-11 with biocontrol effects and its application. The fermented Pichia pastoris LZ-11 strain was isolated from plum fruits in the orchard of Huazhong Agricultural University in Wuhan, Hubei Province. The volatile gases (VOCs) it produces have a significant inhibitory effect on the mycelial growth and spore germination of sour rot fungi. After being treated with volatile gases from the fermented Pichia pastoris LZ-11 strain, the occurrence of sour rot in Wenzhou mandarin oranges can be completely inhibited, and the prevention and control effect is significant; in addition, the prevention and control effect of the fermented Pichia pastoris LZ-11 strain on citrus green mold disease reaches 76%, providing a new means for safe and effective prevention and control of plant diseases.

[0004] Therefore, screening biocontrol microorganisms with good antibacterial effects is the basis for developing biological control agents. It is also of great significance to enrich the resources of biocontrol microorganisms and the prevention and control of fruit tree diseases and other diseases. Summary of the invention

[0005] In order to solve the above problems, the present invention proposes a strain of Pichia kudriavzevii and application of its volatile substances.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention proposes a Pichia kudriavzevii Bg7, whose classification name is Pichiakudriavzevii, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with a deposit number of CGMCC No. 32277, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] In a second aspect, the method for culturing the above-mentioned Pichia kudriavzevii Bg7 is to inoculate the Pichia kudriavzevii Bg7 on a YPDA medium at a culture temperature of 25-30°C.

[0009] In a third aspect, the present invention further provides a biocontrol agent, which comprises a bacterial solution of the above-mentioned Pichia kudriavzevii Bg7 and / or a volatile substance with antibacterial activity produced by the bacterial solution.

[0010] Preferably, the volatile substances are esters, acids, alcohols, ketones and aldehydes.

[0011] Preferably, the above-mentioned volatile substances include isoamyl acetate, ethyl propionate, ethyl butyrate, isoamyl propionate, ethyl hexanoate, ethyl octanoate, ethyl isovalerate, isobutyl acetate, ethyl isobutyrate, ethyl acetate, 2-methylpropionic acid, propionic acid, acetic acid, isovaleric acid, isoamyl alcohol, cis-2-penten-1-ol, isobutanol, acetophenone, 3-hydroxy-2-butanone, phenylacetaldehyde and n-hexanal.

[0012] Preferably, the bacterial solution of Pichia kudriavzevii Bg7 is obtained by inoculating Pichia kudriavzevii Bg7 into YPDB liquid culture medium and culturing at 25-30° C. and 180 r / min for 12-16 h.

[0013] A fourth aspect is the use of the above-mentioned biocontrol agent in inhibiting the growth of pathogenic fungi.

[0014] Preferably, the above-mentioned pathogenic fungi are any one or more of pear rot pathogen, strawberry gray mold pathogen and apple ring rot pathogen.

[0015] A fifth aspect is the use of the above-mentioned biocontrol agents in preventing and controlling plant diseases.

[0016] Preferably, the plant diseases are any one or more of pear rot, strawberry gray mold and apple ring rot. The present invention has the following beneficial effects:

[0017] The present invention has discovered a strain of Pichia kudriavzevii Bg7 with biological control effect. Through plate confrontation experiment, it is found that strain Bg7 strongly inhibits the growth of pear rot pathogen, and the inhibition rate is 58.16%. By using the counter-plate method, the culture medium coated with Bg7 bacterial solution is counter-plated with the pathogen culture medium, and it is found that the volatile organic compounds produced by Bg7 significantly inhibit the growth of pear rot pathogen, strawberry gray mold pathogen and apple ring rot pathogen, and the inhibition rates are 64.16%, 100% and 59. 92%; the pear tree rot disease inoculated with Bg7 plate was cultured side by side, and 7 days after inoculation, the lesions on the branches treated with volatile organic compounds of strain Bg7 were all 0 cm; the strawberry fruit inoculated with strawberry gray mold disease was cultured side by side with Bg7 plate, and 3 and 4 days after inoculation, the lesions on the samples treated with volatile organic compounds of strain Bg7 were also 0 cm, indicating that the volatile organic compounds produced by strain Bg7 can completely inhibit the occurrence of pear tree rot disease and strawberry gray mold disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a phylogenetic tree constructed based on 26S rDNA sequences.

[0020] Figure 2 This is the inhibition of strain Bg7 on pear rot pathogen; the left picture is the control pear rot pathogen, and the right picture is the co-culture of strain Bg7 and pear rot pathogen.

[0021] Figure 3 This is the inhibition of volatile organic compounds of strain Bg7 on different pathogens.

[0022] Figure 4 The control effect of volatile organic compounds of strain Bg7 on pear tree rot disease.

[0023] Figure 5 The control effect of volatile organic compounds of strain Bg7 on strawberry gray mold. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0026] The present invention proposes a Pichia kudriavzevii Bg7, whose classification name is Pichiakudriavzevii, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with a deposit number of CGMCC No. 32277, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0027] Example 1

[0028] Isolation of strains:

[0029] The invention separates endophytes from strawberry leaves in an experimental field of Zhengzhou Fruit Research Institute of Chinese Academy of Agricultural Sciences in Zhengzhou, Henan Province. One of the strains is identified as Pichia kudriavzevii of the genus Pichia by morphology and molecular biology and is named Bg7.

[0030] The strain isolation method is as follows: select a 5×5mm strawberry leaf tissue block, disinfect it with 75% ethanol for 1min, then disinfect it with 1% NaClO for 5min, then wash it with sterile water 3 times, dry the water on the surface of the tissue block with sterilized filter paper, place the tissue block on YPDA medium (containing 100mg / L kanamycin sulfate), and culture it in an incubator at 25°C, and observe it every 24h. After the colony is formed, pick strains with different colony morphologies and inoculate them on new YPDA medium, and separate and purify them to obtain pure strains, one of which is named Bg7.

[0031] Pick a single colony of strain Bg7 and add it to YPDB liquid medium for culture to obtain a fresh culture solution of Bg7. Take 4 mL of fresh culture solution and centrifuge at 4°C and 10000 rpm for 5 min, collect the bacteria, and extract DNA using a DNA extraction kit. After electrophoresis detection, use the DNA of strain Bg7 as a template and primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') for PCR amplification to amplify the 26s rDNA sequence of strain Bg7 and obtain a fragment with a length of 570 bp, the sequence of which is shown in SEQ ID No. 1.

[0032] The 26s rDNA sequence of strain Bg7 was compared with the sequence similarity of Pichia kudriavzevii on the NCBI website. The highest similarity between the gene sequence and Pichia kudriavzevii was 100%. Phylogenetic tree analysis showed that strain Bg7 and two P. kudriavzevii strains were in the same branch ( Figure 1 ). These results indicate that strain Bg7 belongs to the genus Pichia kudriavzevii.

[0033] Example 2

[0034] The plate confrontation experiment was used to detect the inhibition of the isolated and purified strain Bg7 on the pear rot pathogen:

[0035] The pathogen of pear rot was inoculated on a PDA plate and cultured at 25°C for 4 days. A 5mm diameter puncher was used to punch out the bacterial block with consistent colony growth and inoculated it in the center of the PDA plate. The isolated and purified strain Bg7 was inoculated 2 cm above, below, left and right of the pathogen. The pathogen alone was used as the control. Four plates were inoculated for each treatment and cultured at a constant temperature of 25°C. Three replicates were performed. After the pathogen in the control group grew all over the culture plate, the antibacterial rate was calculated.

[0036] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control plate colony diameter × 100%.

[0037] The results showed that strain Bg7 showed a strong inhibitory effect on pear rot pathogens ( Figure 2 ), the inhibition rate was 58.16%.

[0038] Example 3

[0039] Inhibition of volatile organic compounds of strain Bg7 on different pathogenic fungi:

[0040] Pick a single colony of strain Bg7, add it to YPDB liquid medium, and culture it at 25°C, 180r / min for 16h to obtain a fresh culture solution of Bg7 (OD600=0.8-1.0). Pipette 100 μL of Bg7 culture solution and evenly spread it on YPDA medium, and culture it at 25°C for 24h.

[0041] The double-stitch method was used. A 0.5 cm diameter puncher was used to punch bacterial cakes at the edge of the colonies of the pathogens of pear rot, strawberry gray mold, and apple ring rot. The cakes were placed in the center of the PDA plates, and they were all placed on the plates inoculated with the strain Bg7. The plates were sealed with sealing film. The YPDA plates not coated with the test strain Bg7 were used as blank controls. The plates were cultured at a constant temperature of 25°C, and each treatment was repeated three times. The diameter of the pathogen colony (cm) was measured 4 days later, and the inhibition rate was calculated.

[0042] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control plate colony diameter × 100%.

[0043] The results showed that the volatile organic compounds of strain Bg7 had significant inhibitory effects on pear rot pathogen, strawberry gray mold pathogen, and apple ring rot pathogen ( Figure 3 ), the inhibition rates were 64.16%, 100% and 59.92% respectively.

[0044] Example 4

[0045] The control effect of volatile organic compounds of strain Bg7 on pear rot:

[0046] Healthy one-year-old pear branches were selected, and after surface disinfection, they were cut into 7 cm long segments and placed in a sterile culture dish with a diameter of 9 cm. A hole was punched in the middle of the branch with a 0.5 cm diameter puncher, and a pear rot pathogen cake of the same diameter was placed on the hole. It was then placed on a plate (9 cm in diameter) of strain Bg7 cultured on a YPDA plate for 24 hours, sealed with a sealing film, and an empty YPDA plate was used as a blank control. The plate was placed in a 25°C light incubator for culture, and the disease was observed after 7 days, and the diameter of the lesions was measured. The results showed that the volatile organic compounds of strain Bg7 could completely inhibit the occurrence of pear rot ( Figure 4 A). The statistical results showed that the lesions on the CK branches were 3.20 cm, while the lesions on the branches treated with volatile organic compounds of strain Bg7 were all 0 cm ( Figure 4 B).

[0047] Example 5

[0048] The control effect of volatile organic compounds of strain Bg7 on strawberry gray mold:

[0049] Healthy strawberry fruits of uniform size were selected, and after surface disinfection, strawberry gray mold cakes (0.5 cm in diameter) were inoculated. Six fruits were inoculated for each treatment, and the treated fruits were placed in a 15 cm × 2.5 cm round culture dish, and matched with a plate of strain Bg7 (15 cm × 2.5 cm) cultured on a YPDA plate for 24 hours. The plates were sealed with sealing film, and an empty YPDA plate was used as a blank control. The plates were cultured in a 25°C light incubator, and the disease was observed after 3 and 4 days, and the diameter of the lesions was measured. The results showed that the volatile organic compounds of strain Bg7 could completely inhibit the occurrence of strawberry gray mold ( Figure 5 A). After 3 and 4 days of inoculation, the lesions on the control strawberry fruits were 1.80 cm and 2.19 cm respectively, while the lesions on the samples treated with volatile organic compounds of strain Bg7 were 0 cm ( Figure 5 B).

[0050] Example 6

[0051] Collection and identification of volatiles from strain Bg7

[0052] The volatiles of strain Bg7 were determined using headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) technology, and the steps are as follows:

[0053] 1) Sample processing method: The yeast sample inoculated in a 20 mL headspace bottle was incubated at 60°C for 15 min before injection, and each sample was measured in triplicate.

[0054] 2) Headspace injection conditions: incubation temperature of 60°C; incubation time of 15 min; injection volume of 500 μL; splitless injection; incubation speed of 500 r / min; injection needle temperature of 85°C.

[0055] 3) GC conditions: chromatographic column temperature is 60°C; carrier gas: high-purity helium (purity ≥ 99.999%); programmed pressure increase: initial flow rate 2.00 mL / min, maintained for 2 min, linearly increased to 10.00 mL / min within 8 min, linearly increased to 100.00 mL / min within 10 min, maintained for 30 min. Chromatographic run time: 30 min; injection port temperature: 80°C.

[0056] 4) Ionization source: tritium source (3H); migration tube length: 53 mm; electric field strength: 500 V / cm; migration tube temperature: 45°C; drift gas: high-purity nitrogen (purity ≥ 99.999%); flow rate: 150 mL / min; positive ion mode.

[0057] 5) Data processing: The retention index of the substance is then calculated based on the retention time of the target substance, and the GC retention index (NIST 2020) database and IMS migration time database built into the VOCal software are used for retrieval and comparison to perform a qualitative analysis of the target substance.

[0058] Table 1 Volatile components of strain Bg7

[0059]

[0060] Excluding the blank control treatment, strain Bg7 produced 21 major volatile substances, including esters, acids, alcohols, ketones and aldehydes (Table 1). Among them, ester substances are the most numerous and have a higher content, with a total of 10 substances, namely isoamyl propanoate (3-methylbutyl propanoate), isoamyl acetate (1-Butanol, 3-methyl-, acetate), ethyl caproate (Ethyl caproate), ethyl octanoate (Ethyl octanoate), ethyl isovalerate (Butanoic acid id 3-methylethyl ester), ethyl butanoic acid ethyl ester (Butanoic acid ethyl ester), isobutyl acetate (2-methyl-1-propylacetate), ethyl isobutyrate (Ethyl2-methylpropionate), ethyl propanoate (Ethyl propanoate), and ethyl acetate (Acetic acid ethyl ester); followed by acids, a total of 4 substances, including 2-Methyl propanoic acid (2-Methyl propanoic acid), propanoic acid (Propanoic acid), acetic acid (Acetic acid), and isovalerate (3-Methylbutanoic acid); there are 3 alcohol substances, 2 ketones and 2 aldehyde substances each. Further analysis showed that isoamyl acetate was the highest content, followed by ethyl propionate and ethyl butyrate.

[0061] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Pichia kudriavzevii Bg7, whose taxonomic name is Pichia kudriavzevii , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with the deposit number CGMCC No.32277, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The method for culturing Pichia kudriavzevii Bg7 according to claim 1, characterized in that: Pichia kudriavzevii Bg7 was inoculated on YPDA medium at a culture temperature of 25-30°C.

3. A biocontrol agent, characterized in that: The biocontrol agent comprises the bacterial solution of Pichia kudriavzevii Bg7 according to claim 1 and / or a volatile substance with antibacterial activity produced by the bacterial solution.

4. The biocontrol agent according to claim 3, characterized in that: The volatile substances include isoamyl acetate, ethyl propionate and ethyl butyrate.

5. The biocontrol agent according to claim 4, characterized in that: The volatile substances also include isoamyl propionate, ethyl hexanoate, ethyl octanoate, ethyl isovalerate, isobutyl acetate, ethyl isobutyrate, ethyl acetate, 2-methylpropionic acid, propionic acid, acetic acid, isovaleric acid, isopentanol, cis-2-penten-1-ol, isobutanol, acetophenone, 3-hydroxy-2-butanone, phenylacetaldehyde and n-hexanal.

6. The biocontrol agent according to claim 3, characterized in that: The bacterial liquid is obtained by inoculating Pichia kudriavzevii Bg7 into a YPDB liquid culture medium and culturing at 25-30° C. and 180 r / min for 12-16 hours.

7. Use of the biocontrol agent according to any one of claims 3 to 6 in inhibiting the growth of pathogenic bacteria and fungi.

8. The use according to claim 7, characterized in that: The pathogenic fungi are any one or more of pear rot pathogen, strawberry gray mold pathogen and apple ring rot pathogen.

9. Use of the biocontrol agent according to any one of claims 3 to 6 in preventing and controlling plant diseases.

10. The use according to claim 9, characterized in that: The plant diseases are any one or more of pear rot, strawberry gray mold and apple ring rot.

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