CN113773966B - Alternaria tenuissima and application thereof - Google Patents

Abstract

The invention relates to Alternaria tenuissima and application thereof, and belongs to the technical field of microorganisms. The Alternaria tenuissima (Alternaria tenuissima) XL-0016 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23047 in the year 7 and the day 15 of 2021. The strain has obvious inhibition effect on 5 crop pathogenic fungi, namely sugarcane black spot pathogen, potato fusarium wilt pathogen, rice sheath blight pathogen, wheat gibberella, corn big spot pathogen, and has wide application prospect and easy popularization and application.

Description

Alternaria tenuissima and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to Alternaria tenuissima and application thereof in bacteriostasis.

Background

Alternaria tenuissima (Alternaria tenuissima) is a saprophytic filamentous conditional pathogenic bacterium widely existing in nature and belonging to the genus Alternaria of the order Alternaria, the family Heimycetes. Alternaria minutissima is not only an important plant pathogenic fungus, but also a biological resource with application prospect, and has wide application prospect in aspects of biological control, biological herbicide, environmental waste treatment, variety selection and the like.

Sugarcane black spot is a worldwide disease caused by black powder fungus (Ustilago scitaminea Syd.) and is also the most dominant fungal disease that jeopardizes sugarcane production in China. Black spot disease commonly occurs in various sugarcane planting provinces in China, is more serious especially on dry-land sugarcane and perennial root sugarcane, has the incidence rate of up to 80% -90% in certain sugarcane fields, and seriously affects the sustainable development of sugarcane industry.

Potato wilt is a fungal soil-borne vascular bundle disease caused by fusarium oxysporum (Fusarium oxysporum) and is widely distributed, and occurs to different degrees in various potato planting areas in China. In recent years, as the planting area of the potatoes increases year by year, continuous cropping is more and more serious, so that the disease resistance of the potatoes is reduced, the occurrence of potato wilt is more and more serious, generally about 30% of yield is reduced, 78% of plants die when serious, the yield and commodity of the potatoes are directly reduced, and the sustainable development of the potato industry is severely restricted.

Rice sheath blight is one of three fungal diseases of rice, the harm of the fungal diseases is distributed in all rice areas worldwide, the rice yield is seriously influenced each year, more than 50% of yield reduction can be caused in fields with serious disease, and the fungal diseases are serious restriction factors for high yield and stable yield of rice. The pathogen of the disease is rhizoctonia solani (Rhizoctonia solani K uhn), can cause serious diseases on various economic crops, and has important influence on the yield and quality of the crops.

Wheat scab is caused by fungus Fusarium graminearum (Fusarium graminearum), which not only causes great yield reduction of wheat, but also generates mycotoxin, thus causing great loss to agricultural production and ecology. Due to factors such as precipitation increase, straw returning and the like, wheat scab frequently occurs in large areas in various major wheat producing areas in China, and the wheat scab becomes a major disease of wheat in Huang-Huai wheat areas, and seriously affects the yield and quality of the wheat.

The corn leaf spot disease is a serious leaf disease of harmful corn and is caused by fungus corn leaf spot bacteria. The non-behavior (Exserohilum turcicum) of the corn big spot germ is corn big spot helminth fungus belonging to asexual fungus helminth fungus genus; the sexual form (Setosphaeria turcica) is the Cavity of the large-spot bristle holder, belonging to the genus Ascomycota. This disease occurs widely in cold corn planting areas in america, europe, asia and oceans, and sometimes causes other diseases such as corn root rot, stem rot, etc. The occurrence and prevalence of corn northern leaf blight not only causes serious economic loss but also reduces the quality of corn. The disease is mainly distributed in cold corn planting areas such as northern areas, high-altitude mountain areas and the like in China since 70 s. Generally, the year yield is reduced by about 20%, and in the serious epidemic year and region, the corn yield is reduced by 50% or more.

Of the plant diseases, 70% -80% of the diseases are caused by pathogenic fungal infections. The method is suitable for the national times of food safety production and stable society. The pathogenic fungi disease of crops not only directly causes the yield and the quality of the crops to be reduced, but also can secrete and produce various toxins and metabolites harmful to people and livestock in the process of infecting the crops by part of pathogenic fungi, thus greatly threatening the safety of agricultural products. Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field of microorganisms at present.

Disclosure of Invention

The invention aims to solve the defects of the prior art and provide Alternaria tenuis, which has the characteristics of easiness in culture, high yield, stable culture property and the like, and the culture of Alternaria tenuis has higher antibacterial activity on the 5 crop pathogenic fungi.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the invention provides Alternaria tenuissima (Alternaria tenuissima) XL-0016 which has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23047 in the year 7 and 15 of 2021.

The second aspect of the invention provides the application of Alternaria tenuissima (Alternaria tenuissima) XL-0016 in preparation of a bacteriostatic agent.

Further, preferably, the fungus inhibited by the bacteriostatic agent is sugarcane black spot pathogen, potato fusarium wilt pathogen, rice sheath blight pathogen, wheat scab pathogen or corn big spot pathogen.

Further, it is preferred that the active ingredient in the bacteriostatic agent comprises the Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture.

Further, it is preferable that the Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture is a liquid culture; the culture method comprises the following steps: streaking Alternaria minutissima (Alternaria tenuissima) XL-0016 on PDA slant culture medium, and culturing in a 28 deg.C incubator for 3-5 days;

preparing a PDB liquid culture medium, directly inoculating the grown inclined plane into the PDB liquid culture medium, placing the PDB liquid culture medium into a shaking table at 28 ℃ and 180rpm for culturing for 15-20 days, and taking out the PDB liquid culture medium to obtain a liquid culture;

the PDA slant culture medium comprises the following formula: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2;

the formula of the PDB liquid culture medium comprises the following components: potato extract powder 5g/L, glucose 15g/L, peptone 10g/L and sodium chloride 5g/L.

In a third aspect the invention provides said Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture.

The fourth aspect of the invention provides the use of Alternaria tenuissima (Alternaria tenuissima) XL-0016 in the preparation of a bacteriostatic agent, wherein the active ingredient in the bacteriostatic agent comprises an extract of Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture; the extract is obtained by taking Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture as a raw material and extracting the raw material by an organic solvent.

Further, it is preferable that the organic solvent is ethyl acetate.

In a fifth aspect the invention provides an extract of a culture of Alternaria tenuissima (Alternaria tenuissima) XL-0016.

The Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture can also be obtained by taking Alternaria tenuissima (Alternaria tenuissima) XL-0016 as a raw material and culturing the Alternaria tenuissima by a PDA culture medium, a PDB culture medium and the like which are conventional in the art.

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

the invention provides Alternaria tenuissima and application thereof, and the liquid culture of Alternaria tenuissima XL-0016 has remarkable inhibition effect on 5 fungi of sugarcane black spot pathogen (Ustilago scitaminea Syd., NB-01), potato wilt pathogen (Fusarium oxysporum Schlecht, NB-02), rhizoctonia solani (Thanatephorus cucumeris, NB-04), wheat scab pathogen (Fusarium graminearum, NB-05) and corn big spot pathogen (Setosphaeria turcica, NB-06), and has good application prospect.

Drawings

FIG. 1 is a single colony morphology (PDA plate) of Alternaria tenuissima XL-0016; wherein, FIG. 1-A is a front view of a flat panel; FIG. 1-B is a back view of a flat panel;

FIG. 2 shows the inhibition of 5 crop pathogenic fungi by Alternaria tenuissima XL-0016 (PDA plate); wherein, FIG. 2-A is a front view of a flat panel; FIG. 2-B is a back view of the plate;

alternaria tenuissima (Alternaria tenuissima) XL-0016 is preserved in China general microbiological culture Collection center (CGMCC) on the 7 th month 15 day 2021, with the preservation number of CGMCC No.23047 and the preservation address of North Chen West Lu No.1, 3 in the Chaoyang area of Beijing, and the microbiological institute of China academy of sciences.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The materials or equipment used are conventional products available from commercial sources, not identified to the manufacturer.

EXAMPLE 1 Source and identification of Alternaria tenuissima (Alternaria tenuissima) XL-0016

(1) Alternaria tenuissima (Alternaria tenuissima) XL-0016 source

A fungus, designated XL-0016, was isolated from Notoginseng radix picked from Wenshan in Yunnan province on 7.5.2019.

(2) Identification of Alternaria tenuissima (Alternaria tenuissima) XL-0016

The ITS sequencing Sequence of XL-0016 is shown in a Sequence table (SEQ ID NO. 1), and has the highest Sequence similarity with Sequence ID MZ541977.1, and the similarity is 99.46%.

XL-0016 belongs to Alternaria tenuissima (Alternaria tenuissima) according to the above identification.

EXAMPLE 2 principal morphological characteristics of Alternaria tenuissima (Alternaria tenuissima) XL-0016

Inoculating Alternaria tenuissima XL-0016 on potato glucose culture medium, culturing at 28deg.C for 7 days, wherein colony diameter is 80-83mm, the edge of colony is white, the middle part is black, tiling is performed, and hyphae are rare; the quality is granular, the spore stalk bundles are produced in a large quantity, the length is about 2-5mm, the mycelium is white, the back is white, the conidiophores are upright or slightly bent, the color is deep and short, the conidiophores are usually single, the shape of the conidiophores is quite different, the conidiophores are in an inverted stick shape to an oblong shape, and the mature spores have 4-7 diaphragm or semi-diaphragm and 1-6 longitudinal and oblique diaphragms. There are often 1-4 main diaphragms, the sporocysts 15-47.5um x 6.25-12.5um, average 25.25um x 0.5um.

The PDA culture medium formula comprises the following components: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.

The single colony morphology diagram of the strain on a PDA flat plate is shown in figure 1, and figure 1-A is a front view of the flat plate in figure 1-A; FIG. 1-B is a back view of the plate.

EXAMPLE 3 determination of the antibacterial Activity of Alternaria tenuissima (Alternaria tenuissima) XL-0016 against pathogenic fungi

The bacteriostatic ability of fungi to be screened (Alternaria tenuissima XL-0016) to test fungi (ten pathogenic fungi) is measured by adopting a plate counter method, and the specific method is as follows:

PDA plates are prepared, fungi to be screened and fungi to be tested are respectively cultured, after the diameter of a colony reaches 70-80mm, a sterilized puncher with the diameter of 5mm is used for punching holes (equidistant from the edge of a dish) at the positions where the two culture mediums grow consistently. On the PDA blank plate, the perforated culture medium blocks are picked up by sterilized forceps respectively, two fungi are inoculated in reverse directions respectively at a position about 3cm on the same straight line from the center of the plate, and then the size of a bacteriostasis zone is measured every 24 hours (crisscross method). The bacteriostasis rate was calculated according to the following formula:

antibacterial ratio = [ (control colony diameter-5) - (treated colony diameter-5) ] ≡ (control colony diameter-5) ×100%

Note that: "5" is the diameter of the colony taken by the punch.

The PDA culture medium formula comprises the following components: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.

The inhibition rates of Alternaria tenuissima XL-0016 against different pathogenic fungi are shown in Table 1.

TABLE 1 inhibition of Alternaria tenuissima XL-0016 against different pathogenic fungi

Wherein NB-01 is sugarcane black spot germ (Ustilago scitaminea Syd.), NB-02 is potato blight germ (Fusarium oxysporum Schlecht), NB-04 is Rhizoctonia solani (Thanatephorus cucumeris), NB-05 is wheat gibberella (Fusarium graminearum), and NB-06 is corn big spot germ (Setosphaeria turcica).

As can be seen from Table 1, alternaria tenuissima (Alternaria tenuissima) XL-0016 has a high bacteriostatic activity against 5 crop pathogenic fungi, wherein the highest bacteriostatic rate against sugarcane black spot pathogen (Ustilago scitaminea Syd., NB-01) is 53.33%.

The inhibition effect of the strain on 5 crop pathogenic fungi on a PDA flat plate is shown in figure 2, wherein the upper half part of the flat plate is 5 crop pathogenic fungi, and the lower half part of the flat plate is Alternaria tenuis XL-0016, wherein figure 2-A is a front view of the flat plate; FIG. 2-B is a back view of the plate. As can be seen from FIG. 2, alternaria tenuissima XL-0016 shows a significant inhibition of 5 crop pathogenic fungi.

EXAMPLE 4 liquid culture of Alternaria tenuissima (Alternaria tenuissima) XL-0016

Activation of the strain: inoculating the preserved Alternaria tenuissima XL-0016 on PDA slant culture medium by streaking, and culturing in a 28 ℃ incubator for 3-5 days;

liquid culture of strains: preparing 200 ml/bottle PDB liquid culture medium, directly inoculating the grown slant into liquid shake flask, culturing in shake flask at 28deg.C and 180rpm for 15-20 days, and taking out to obtain liquid culture (i.e. fermentation broth).

The PDA slant culture medium comprises the following formula: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.

The formula of the PDB liquid culture medium comprises the following components: potato soaked powder 5g/L, glucose 15g/L, peptone 10g/L, sodium chloride 5g/L, and pH value is natural.

EXAMPLE 5 preparation of Alternaria tenuissima (Alternaria tenuissima) XL-0016 Ethyl acetate crude extract

The fermentation broth of example 4 was extracted with an equal volume of ethyl acetate by: oscillating for 30min, fully mixing the fermentation liquor with ethyl acetate, standing for 24h to separate liquid, taking out ethyl acetate phase, namely an upper phase, performing reduced pressure rotary evaporation at 42 ℃ (the vacuum degree is generally-0.08 Mpa to-0.01 Mpa), completely evaporating the solvent by using a rotary evaporator to obtain bacterial liquid ethyl acetate crude extract, and preserving in a refrigerator at 4 ℃ for later use.

EXAMPLE 6 determination of MIC and MBC values of Alternaria tenuissima (Alternaria tenuissima) XL-0016 ethyl acetate crude extract

The bacterial liquid ethyl acetate crude extract of example 5 was further measured for Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using a 96-well plate method, as follows:

(1) culturing a test bacterial liquid: and (3) streaking and inoculating the preserved test fungi (5 pathogenic fungi) on a PDA slant culture medium, placing the PDA slant culture medium in a 28 ℃ incubator for 3-5 days, directly inoculating the grown slant into a standby PDB liquid culture medium, placing the PDB liquid culture medium in a shaking table at 28 ℃ and 180rpm for culturing for 48 hours, and taking out the liquid culture medium for standby, thus obtaining the test fungus liquid.

(2) Sample solution preparation: 1mg of the crude ethyl acetate extract of the bacterial liquid of example 5 was taken, and 100uL of DMSO was added to dissolve it sufficiently, and the volume was determined by DMSO to obtain a sample solution of 100 ug/mL.

(3) MIC and MBC were determined in 96-well plate method: taking a sterile 96-well plate, adding 100 mu L of PDB liquid culture medium into 1-7 wells, adding 100 mu L of sample solution into 1-well, mixing uniformly (the concentration is 50 ug/mL), taking 100 mu L out of 1-well, adding into 2-well, mixing uniformly, diluting to 7-well in sequence, taking 100 mu L out of 7-well, and discarding. Sample concentrations in wells 1-7 were 50, 25, 12.5, 6.25, 3.125, 1.563 and 0.7815ug/mL, respectively, with 100. Mu.L of the test bacteria solution. On the same 96-well plate, a well with 100. Mu.L of DMSO was used as a blank control, a well with no sample solution but 100. Mu.L of the test bacterial liquid was used as a positive control, and a well with no test bacterial liquid but 100. Mu.L of PDB liquid medium was used as a negative control. The minimum concentration of clarified culture solution in the observation wells is MIC, 50. Mu.L of clarified liquid in the small wells is coated on PDA culture medium, and the culture is inverted at 28 ℃ for 48 hours and repeated three times, and the minimum concentration of no bacteria growth on the PDA culture medium is MBC.

The PDA culture medium formula comprises the following components: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2.

The formula of the PDB liquid culture medium comprises the following components: potato soaked powder 5g/L, glucose 15g/L, peptone 10g/L, sodium chloride 5g/L, and pH value is natural.

The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Alternaria tenuissima XL-0016 against different pathogenic fungi are shown in Table 3.

TABLE 2 Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Alternaria tenuissima XL-0016 against different pathogenic fungi

As can be seen from Table 2, the ethyl acetate crude extract of XL-0016 liquid culture has remarkable inhibition effect on 5 crop pathogenic fungi, namely, sugarcane black spot, potato fusarium, banded sclerotial blight, wheat gibberella and corn big spot.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Yang Ling future Environment protection technology Co., ltd

<120> Alternaria minutissima and use thereof

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<170> SIPOSequenceListing 1.0

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<212> DNA

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Claims (3)

1. Alternaria tenuissima (Alternaria tenuissima) XL-0016 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 23047 in the year 7 and the day 15 of 2021.

2. The application of Alternaria tenuissima (Alternaria tenuissima) XL-0016 in preparation of a bacteriostatic agent, which is characterized in that fungi inhibited by the bacteriostatic agent are sugarcane black spot, potato fusarium wilt, rice sheath blight germ, wheat scab germ or corn big spot germ;

the active ingredients in the bacteriostat comprise extracts of Alternaria tenuissima (Alternaria tenuissima) XL-0016 cultures; the extract is obtained by taking Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture as a raw material and extracting with an organic solvent;

the organic solvent is ethyl acetate.

3. The use of Alternaria tenuissima (Alternaria tenuissima) XL-0016 according to claim 2 for the preparation of a bacteriostatic agent, wherein the Alternaria tenuissima (Alternaria tenuissima) XL-0016 culture is a liquid culture; the culture method comprises the following steps: streaking Alternaria minutissima (Alternaria tenuissima) XL-0016 on PDA slant culture medium, and culturing in a 28 deg.C incubator for 3-5 days;

preparing a PDB liquid culture medium, directly inoculating the grown inclined plane into the PDB liquid culture medium, placing the PDB liquid culture medium into a shaking table at 28 ℃ and 180rpm for culturing for 15-20 days, and taking out the PDB liquid culture medium to obtain a liquid culture;

the PDA slant culture medium comprises the following formula: 3g/L of potato soaked powder, 20g/L of glucose, 14g/L of agar and pH value of 5.6+/-0.2;

the formula of the PDB liquid culture medium comprises the following components: potato extract powder 5g/L, glucose 15g/L, peptone 10g/L and sodium chloride 5g/L.