阅读说明：本技术 一株具有抑菌作用的葡萄汁有孢汉逊酵母mp1861及其应用 (Hansenula polymorpha MP1861 with bacteriostatic effect on grape juice and application thereof ) 是由 王光远 刘子瑜 张晓峰 梁文星 李德龙 李敬涛 于 2021-06-16 设计创作，主要内容包括：本发明公开了一株具有抑菌作用的葡萄汁有孢汉逊酵母MP1861及其应用。所述葡萄汁有孢汉逊酵母MP1861的分类命名为葡萄汁有孢汉逊酵母Hanseniaspora uvarum,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO：M 2021613；其26S rDNA的核苷酸序列如SEQ ID No.3所示。本发明经过抑菌实验证实,葡萄汁有孢汉逊酵母MP1861对大豆疫霉、烟草疫霉、辣椒疫霉、茄科雷尔氏菌均具有显著的抑制作用。因此,葡萄汁有孢汉逊酵母MP1861及其发酵滤液能够用于制备植物病原菌抑制菌以及防治疫霉病和/或青枯病的生防菌剂,其使用安全,具有良好的市场应用前景。(The invention discloses a Hansenula polymorpha MP1861 strain with an antibacterial effect on grape juice and application thereof. The classification of Hansenula polymorpha MP1861 into Hansenula polymorpha Hanseniaspora uvarum And the culture is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021613; the nucleotide sequence of 26S rDNA is shown in SEQ ID No. 3. The bacteriostatic experiment proves that the Hansenula polymorpha MP1861 contained in the grape juice has obvious inhibition effect on phytophthora sojae, phytophthora nicotianae, phytophthora capsici and ralstonia solanacearum. Thus, grape juiceThe hansenula polymorpha MP1861 and the fermentation filtrate thereof can be used for preparing plant pathogenic bacteria inhibiting bacteria and biocontrol microbial inoculum for preventing and treating phytophthora blight and/or bacterial wilt, are safe to use, and have good market application prospect.)

1. A Hansenula polymorpha MP1861 strain with bacteriostasis function is characterized in that: it is classified and named as Hansenula polymorpha of grape juiceHanseniaspora uvarumAnd the culture is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021613.

2. The Hansenula polymorpha MP1861 as claimed in claim 1, wherein: the nucleotide sequence of the 26S rDNA of the Hansenula polymorpha MP1861 is shown as SEQ ID No. 3.

3. The Hansenula polymorpha MP1861 as claimed in claim 1, wherein: the colony of the Hansenula polymorpha MP1861 in the grape juice is round, milky, smooth in edge, wet in surface, opaque and convex in the middle.

4. Use of Hansenula polymorpha MP1861 as claimed in any of claims 1 to 3 for the preparation of a phytopathogen inhibitor.

5. Use according to claim 4, characterized in that: the plant pathogenic bacteria include phytophthora nicotianae, phytophthora capsici, phytophthora sojae and ralstonia solanacearum.

6. Use according to claim 5, characterized in that: the Hansenula polymorpha MP1861 containing grape juice can obviously inhibit the hypha growth, spore germination and sprout tube elongation of phytophthora nicotianae, phytophthora capsici, phytophthora sojae and ralstonia solanacearum.

7. The use of Hansenula polymorpha MP1861 as claimed in any of claims 1-3 for the preparation of biocontrol agents for the control of phytophthora blight and/or bacterial wilt.

8. Use according to claim 7, characterized in that: the biocontrol microbial inoculum contains hansenula polymorpha MP1861 fermentation liquor of grape juice.

9. Use according to claim 8, characterized in that: the volume ratio of the hansenula polymorpha MP1861 fermentation broth in the biocontrol microbial inoculum is 10-30%.

10. Use according to claim 7, characterized in that: the phytophthora root rot is phytophthora root rot of plants caused by phytophthora nicotianae, phytophthora capsici and/or phytophthora sojae; the bacterial wilt is plant bacterial wilt caused by Ralstonia solanacearum.

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a hansenula polymorpha MP1861 with an antibacterial effect on grape juice and application thereof.

Background

At present, agricultural production faces many challenges, wherein plant diseases, environmental pollution, ecological damage and other problems are increasingly prominent, which seriously hinders sustainable development of agriculture, and therefore, agricultural production bears unprecedented enormous pressure. Especially, plant diseases can not only cause the reduction of crop yield, but also seriously threaten the quality safety of agricultural products and international trade thereof to a certain extent. Plant diseases are mainly caused by plant pathogenic bacteria, and the plant pathogenic bacteria are gradually accumulated in the environment, so that the crop diseases are increasingly aggravated, and great economic losses are caused. Among them, phytophthora blight of plants caused by phytophthora and ralstonia solanacearum are relatively common plant diseases.

There are many kinds of phytophthora, among which phytophthora sojae, phytophthora nicotianae, and phytophthora capsici are common phytophthora. Phytophthora sojae causes phytophthora sojae, which is harmful in the whole growth cycle of soybean, causing withering of plants, rotting of roots, and poor development, and is destructive and can cause the soybean to be out of stock. Tobacco black shank caused by phytophthora nicotianae mainly infects roots and stem bases of tobacco to form black concave scabs thereon. The tobacco black shank is all over the world, particularly in temperate, subtropical and tropical regions, and nearly 40 countries and regions are harmed by the tobacco black shank. Phytophthora capsici caused by infection is a common disease occurring in capsicum. Phytophthora capsici has wide host range and strong destructiveness, often harms hundreds of crops of solanaceae, cucurbitaceae, beans and the like, and the caused plant blight is a destructive disease in agricultural production, which causes huge economic loss.

The ralstonia solanacearum is also called ralstonia solanacearum, can cause plant bacterial wilt, and is a common crop pathogenic bacterium. The ralstonia solanacearum can invade from the root or stem base wound of the crop, and can propagate in the vascular bundle after invasion, so that the vascular bundle is browned and rotted, and stem leaves are wilted due to the lack of normal water supply. The host range of the strain is wide, and the strain can infect various crops such as tomatoes, tobaccos and the like, thereby seriously threatening the safety of world agricultural production.

At present, chemical prevention and control means such as agriculture are mainly adopted for controlling plant diseases, but the problems of environmental pollution and drug residue are caused by using a large amount of pesticides for a long time. The development of biological bactericides for biological control is one of the sustainable health trends of agriculture in China.

Disclosure of Invention

The invention aims to provide a hansenula polymorpha MP1861 strain with an antibacterial effect on grape juice and application thereof. The Hansenula polymorpha MP1861 serving as grape juice has the effects of obviously inhibiting phytopathogens of soybean phytophthora, tobacco phytophthora, phytophthora capsici and ralstonia solanacearum, and can be used for preparing biocontrol microbial inoculum.

In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:

the invention provides a Hansenula polymorpha MP1861 with bacteriostatic action on grape juice, which is classified and named as Hansenula polymorpha MP1861Hanseniaspora uvarumAnd the culture is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021613.

Further, the nucleotide sequence of the 26S rDNA of Hansenula polymorpha MP1861 is shown in SEQ ID No. 3.

Furthermore, the colony of Hansenula polymorpha MP1861 in grape juice is round, milky, smooth in edge, moist in surface, opaque and convex in middle.

The invention also provides application of the Hansenula polymorpha MP1861 as a grape juice in preparation of a phytopathogen inhibitor.

Further, the phytopathogens include phytophthora nicotianae, phytophthora capsici, phytophthora sojae, and ralstonia solanacearum.

Further, the hansenula polymorpha MP1861 serving as grape juice can remarkably inhibit the hypha growth, spore germination and bud tube elongation of phytophthora nicotianae, phytophthora capsici, phytophthora sojae and ralstonia solanacearum.

The invention also provides application of the Hansenula polymorpha MP1861 as a grape juice in preparation of a biocontrol microbial inoculum for preventing and treating phytophthora blight and/or bacterial wilt.

Furthermore, the biocontrol microbial inoculum contains hansenula polymorpha MP1861 fermentation liquor of grape juice.

Further, the preparation method of the hansenula polymorpha MP1861 fermentation broth is as follows: hansenula polymorpha MP1861 is inoculated into a potato glucose liquid culture medium, shaking culture is carried out at the temperature of 28 ℃ and the rpm of 160 for 24 h, then centrifugation is carried out at the rpm of 9000 for 10 min, filtration is carried out, and the supernatant is filtered by a bacterial filter with the diameter of 0.22 mu m, thus obtaining the Hansenula polymorpha MP1861 fermentation liquid without thalli.

Furthermore, the volume ratio of the hansenula polymorpha MP1861 fermentation broth in the biocontrol microbial inoculum is 10-30%.

Further, the phytophthora root rot is phytophthora root rot of plants caused by phytophthora nicotianae, phytophthora capsici and/or phytophthora sojae; the bacterial wilt is plant bacterial wilt caused by Ralstonia solanacearum.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the Hansenula polymorpha MP1861 with an inhibiting effect on phytophthora and ralstonia solanacearum is screened from the leaves in the mountains, has a remarkable inhibiting effect on phytophthora nicotianae, phytophthora capsici, phytophthora sojae and ralstonia solanacearum, and specifically is the Hansenula polymorpha MP1861 with the inhibiting effect on hypha growth, spore germination and sprout canal elongation of the phytopathogen, so that the resistance of the plants to phytophthora caused by the phytophthora and plant bacterial wilt caused by the ralstonia solanacearum is effectively improved. Therefore, the grape juice Hansenula polymorpha MP1861 and the fermentation filtrate thereof can be used for preparing bacteriostatic agents or biocontrol microbial agents of plant pathogenic bacteria, and the prepared preparation is simple to use, good in effect, safe and reliable, and has good market application prospect.

Drawings

FIG. 1 is a photograph of a culture plate of Hansenula polymorpha MP 1861;

FIG. 2 shows the result of the inhibition of the growth of phytophthora sojae by Hansenula Bovinifera MP 1861;

FIG. 3 shows the result of the inhibition of the growth of Phytophthora nicotianae hyphae by Hansenula polymorpha MP 1861;

FIG. 4 shows the result of the inhibition of the growth of Phytophthora capsici hyphae by Hansenula botrytis MP 1861;

FIG. 5 shows the results of the experiment of the fermentation filtrate of Hansenula polymorpha MP1861 on the germination of phytophthora nicotianae spores and the elongation of the germ tube;

FIG. 6 is the results of the experiment of the fermentation filtrate of Hansenula polymorpha MP1861 on the germination of phytophthora capsici spores and the elongation of germ tubes;

FIG. 7 shows the results of growth inhibition of Hansenula polymorpha MP1861, a grape juice, against Ralstonia solanacearum.

Detailed Description

The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1: isolation, screening and characterization of strains

First, separation and screening of bacterial strains

Collecting 12 parts of different leaf samples from Laoshan mountain in Qingdao in 7 months in 2018, taking leaves for preliminary examination (removing pathological changes and necrotic parts), and cutting the leaves for later use; inoculating leaf blades into YPD liquid medium (1% Yeast Extract, 2% Peptone, 2% Dextrose) containing chloramphenicol at final concentration of 0.01%, shaking-culturing at 28 deg.C and 180 rpm for 2 d; diluting the culture solution with sterile water to 10 deg.C^-6And (4) carrying out coating separation on YPD plates, and primarily observing yeast strains after culturing for 2 days at 28 ℃. And selecting proper single colonies for streaking separation again to obtain pure culture strains.

Selecting a yeast single colony, respectively inoculating the single colony and 3 kinds of phytophthora (phytophthora sojae, phytophthora nicotianae and phytophthora capsici) to a potato glucose agar (PDA) plate (200 g of potatoes are cleaned and cut into blocks, adding proper water and boiling for 20-30 min, filtering by using gauze to obtain potato juice, adding 20 g of glucose and 20 g of agar, fixing the volume to 1000 ml, and culturing at 26-28 ℃. By testing the inhibition condition of the yeast on the growth of the hyphae of the plant pathogenic bacteria, an MP1861 strain with strong inhibition on the growth of the hyphae of the phytophthora sojae, the phytophthora nicotianae and the phytophthora capsici is screened out.

The plate culture photo of the strain MP1861 is shown in FIG. 1, and the colony is round, milky white, smooth in edge, moist in surface, opaque and convex in middle.

II, classification and identification of MP1861 strain

The MP1861 strain was identified by a 26S rDNA molecular characterization method.

The 26S rDNA sequence primers were: NL 1: GCATATCAATAAGCGGAGGAAAAG (SEQ ID No. 1); NL 4: GGTCCGTGTTTCAAGACGG (SEQ ID No. 2).

The PCR reaction system is as follows: 2 × PCR Master Mix: 12.5 mul; NL 1: 1 mul; NL 4: 1 mul; template: 1 mul; ddH₂O：9.5 μl；

The PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 30 s, annealing at 52 ℃ for 30 s, and extension at 72 ℃ for 0.5 min, and repeating for 30 cycles; renaturation at 72 ℃.

PCR products are recovered by glue, TA cloning is carried out, and single bacteria are picked and sent to a sequencing company for sequencing. The 26S rDNA sequence of the MP1861 strain is shown in SEQ ID No.3 after sequencing.

Compared with NCBI database, MP1861 strain to be detected and Hansenula polymorpha (Hansenula polymorpha) of grape juiceHanseniaspora uvarumstrain B-NC-13-F04) has the sequence similarity of 26S rDNA (GenBank: KJ 794645.1) of 99.82 percent, which indicates that the MP1861 strain is Hansenula polymorpha of grape juiceHanseniaspora uvarum。

The strain preservation is carried out on the Hansenula polymorpha MP1861 strain of the grape juice screened by the invention, and the preservation unit is as follows: china center for type culture Collection; address: wuhan, Wuhan university; the preservation date is as follows:26 months at 2021, grape juice Hansenula polymorphaHanseniaspora uvarumThe preservation number of MP1861 is CCTCC NO: m2021613.

Example 2: biocontrol experiment of Hansenula polymorpha MP1861 on phytophthora (phytophthora sojae, phytophthora nicotianae, phytophthora capsici) in grape juice

1. MP1861 strain inhibiting growth of 3 kinds of phytophthora hyphae

The experimental method comprises the following steps: activating and culturing Hansenula polymorpha MP1861 on YPD plate at 26 deg.C for 2 days; respectively carrying out activated culture on phytophthora nicotianae, phytophthora capsici and phytophthora sojae on a PDA (personal digital assistant) plate at 26-28 ℃ for 3-5 days, punching a circular bacterial cake (the diameter is 0.60 cm) on the edge of activated bacterial hyphae (the growth conditions are as consistent as possible) by using a puncher, respectively picking up the bacterial strain MP1861 and the phytophthora sojae cake to a new plate by using an inoculation needle, taking a blank bacterial cake as a control, then inversely placing a culture dish in an incubator (26-28 ℃) for culture for 4-9 days, measuring the growth conditions of the hyphae, and calculating the inhibition rate of the bacterial strain MP1861 on the growth of three phytophthora sojae hyphaes.

FIGS. 2-4 show the inhibition effect of MP1861 on Phytophthora sojae, Phytophthora nicotianae, and Phytophthora capsici, respectively (Phytophthora in the middle of the figure, MP1861 strain in the four weeks). The result shows that the MP1861 strain has obvious inhibition effect on phytophthora sojae, phytophthora nicotianae and phytophthora capsici. Further analysis shows that the inhibition rate of the MP1861 strain on the growth of the phytophthora sojae is 80%; the hypha growth inhibition rate of the MP1861 strain to phytophthora nicotianae is 65%; the inhibition rate of MP1861 strain on phytophthora capsici hyphae growth was 34%.

2. Inhibition effect of MP1861 fermentation filtrate on phytophthora spore germination and germ tube elongation

The experimental method comprises the following steps: the MP1861 strain is picked up and inoculated into a potato dextrose liquid medium (PDB), shaking culture is carried out at 28 ℃ and 160 rpm for 24 h, then centrifugation is carried out for 10 min under the condition of 9000 rpm, filtration is carried out, and the supernatant is filtered by a bacterial filter with the diameter of 0.22 μm, so as to obtain a fermentation filtrate containing the MP1861 strain fermentation product (the fermentation filtrate does not contain thalli and is only a fermentation product).

Diluting the fermentation filtrate, and detecting by concave glass method (adding sterilized concave glass into the filtrate respectively)Adding 50 μ l of filtrate with different concentrations, wherein the concentration of phytophthora nicotianae spore is 2.4 × 10⁵Per ml, concentration of phytophthora capsici spore is 5.1 × 10⁵Culturing at 26 deg.C for 1 hr, detecting spore germination rate of fermentation filtrate treated with different concentrations, and measuring length of bud tube.

(1) Inhibition of fermentation filtrate of MP1861 strain on phytophthora nicotianae spore germination and germ tube elongation

The result is shown in figure 5, and the inhibition rate of the MP1861 strain on the phytophthora nicotianae spore germination is obviously improved along with the increase of the content of the fermentation filtrate. When the fermentation filtrate reaches 30%, the germination of phytophthora nicotianae spores can be basically inhibited, and the fact that the fermentation product of Hansenula polymorpha MP1861 in grape juice has a strong inhibiting effect on the germination of the phytophthora nicotianae spores is shown. Through measuring the length of the spore germ tube, the inhibition rate of the spore germ tube extension is higher with the increase of the concentration of the filtrate, and the germ tube is basically not extended when the fermentation filtrate reaches 30 percent, which shows that the inhibition rate of the phytophthora nicotianae spore germination and the inhibition rate of the germ tube extension are dose-dependent.

(2) Inhibition of fermentation filtrate of MP1861 strain on phytophthora capsici spore germination and germ tube elongation

The result is shown in figure 6, and the inhibition rate of the spore germination of the phytophthora capsici is obviously improved along with the increase of the content of the fermentation filtrate of the MP1861 strain. When the fermentation filtrate reaches 30%, the germination rate of the phytophthora nicotianae spores is only 7.9%, and the germination rate of the control spores is as high as 97.5%, which shows that the fermentation product of the MP1861 strain has a strong inhibition effect on the germination of the phytophthora nicotianae spores. Through measuring the length of a spore germ tube, the inhibition rate of the spore germ tube extension is higher along with the increase of the concentration of the fermentation filtrate, and the germ tube is basically not extended when the concentration of the fermentation filtrate reaches 30%, which shows that the inhibition rate of the phytophthora capsici spore germination and the inhibition rate of the germ tube extension are dose-dependent.

Example 3: growth inhibition effect of Hansenula polymorpha MP1861 on plant bacterial wilt bacterium solanaceous ralstonia

A sterile cotton swab is used for dipping a liquid of the Ralstonia solanacearum, the liquid is coated on the surface of a plate, and then Hansenula polymorpha MP1861 containing grape juice is inoculated. The culture dish is placed in an incubator (26-28 ℃) in an inverted mode for culturing for 1-2 days, and the bacteriostatic action of the MP1861 strain on the Ralstonia solanacearum is measured. The test result is shown in fig. 7, and an obvious inhibition zone appears around the colonies of the MP1861 strain, which indicates that the Hansenula polymorpha MP1861, a grape juice, has a significant inhibition effect on the growth of the Ralstonia solanacearum.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

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