CN114591866A

CN114591866A - Lactic acid bacteria preparation for preventing and treating cucumber anthracnose and application thereof

Info

Publication number: CN114591866A
Application number: CN202210303898.4A
Authority: CN
Inventors: 贺润晶; 张占忠; 杨雷; 刘爱学; 李建武; 郭朕; 杨博然
Original assignee: Inner Mongolia Zhongfeng Biotechnology Co ltd
Current assignee: Inner Mongolia Zhongfeng Biotechnology Co ltd
Priority date: 2022-03-26
Filing date: 2022-03-26
Publication date: 2022-06-07
Anticipated expiration: 2042-03-26
Also published as: CN114591866B

Abstract

The invention belongs to the technical field of microorganisms, and discloses a compound lactobacillus preparation capable of preventing and treating cucumber anthracnose and application thereof. The active bacteria in the Lactobacillus preparation comprise Lactobacillus plantarum P-8(Lactobacillus plantarum P-8) and Bifidobacterium Lactis V9(Bifidobacterium Lactis V9). After the two bacteria are sprayed on cucumber seedlings with anthracnose as compound bacteria liquid, the mortality of the diseased seedlings is greatly reduced, so that the two bacteria can replace chemical pesticides to prevent and treat the cucumber anthracnose, the disease prevention potential of birth microorganism prevention is better exerted, a foundation is laid for finally planting and cultivating high-quality green cucumber food which is safe to human bodies, and the continuously improved consumption level of consumers is better met.

Description

Lactic acid bacteria preparation for preventing and treating cucumber anthracnose and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent, in particular to a compound lactobacillus preparation capable of preventing and treating cucumber anthracnose and application thereof.

Background

The cucumber has rich nutrition, and has good fragrance and taste. The vegetable is especially suitable for being eaten raw, cold dressed with sauce, cooked food, pickles, salted, sugared, sauced, dried, canned and the like, has special flavor for various eating methods, is a vegetable serving as both fruits and vegetables, and is well popular with the masses. Meanwhile, the cucumber also has the effects of losing weight, beautifying and the like, is generally cultivated all over the country, can be produced all the year round and supplied all the year round, and occupies a very important position in the whole vegetable production.

In 2020, the total production area of global cucumbers is 225 million hectares, and China reaches 127 million hectares, which accounts for 56.4 percent of the whole world. However, anthracnose is an important disease commonly occurring in the south-north vegetable areas in China, most cucumbers occur, plants can be damaged from the seedling stage to the harvest stage, and particularly, the damage of the plants in the later growth stage is the most serious. Because the pathogenic bacteria have strong infectivity and high reproduction rate, once the conditions are proper, the disease can be popularized in a large area, a large number of leaves in the middle and lower parts of the plant are dried, the fruit generates disease spots, the quality is reduced or the commodity value is completely lost, so that the yield of the cucumber is reduced, and the cucumber is even completely harvested in severe cases.

At present, cucumber is cultivated in protected areas and open areas all the year round, and due to the off-season cultivation, the bacterial sources are continuously accumulated, so that the infection cycle of pathogenic bacteria can be continued without interruption. The pathogenic bacteria can form pseudosclerotium in soil for long-term survival, can be planted and saprophytic on a frame material, and can be repeatedly infected as long as the temperature is proper. The anthracnose of cucumber is an important disease of cucumber, and is mainly used for chemical control at present. However, excessive use of pesticides not only causes drug resistance of germs, but also causes high toxicity and high residue of highly toxic pesticides, influences ecological balance to a certain extent, and simultaneously damages the health of people. Therefore, people are more and more concerned about finding efficient pollution-free control methods.

Lactic acid bacteria are fungi which are utilized in large quantities by food microorganisms, and they are widely distributed in nature. A large number of researches show that the lactobacillus can regulate normal flora of gastrointestinal tracts of organisms, keep microecological balance, improve the digestibility and the biovalue of food, reduce serum cholesterol, control endotoxin, inhibit growth and propagation of putrefying bacteria and generation of putrefying products in intestinal tracts, produce nutrient substances and stimulate tissue development, thereby having effects on the nutritional state, physiological functions, cell infection, drug effect, toxic reaction, immune reaction, tumorigenesis, aging process, sudden emergency reaction and the like of the organisms. It is well known that lactic acid bacteria have become commonplace for them to play a practical role in food. With the continuous development of science and technology, lactic acid bacteria are applied more and more. Some lactic acid bacteria are used in dry meat products, are popularized and used due to their own safety, and play a great role.

In addition, some lactic acid bacteria can produce antibacterial substances, and most of the antibacterial spectra are narrow. The bacteriocins produced by the identified lactic acid bacteria are classified into two groups, one group is a narrow antibacterial spectrum lactic acid bacteria having an inhibitory effect only on the relevant bacteria, and the second group is a lactic acid bacteria having a broad spectrum antibacterial activity, which has an inhibitory effect on pathogenic bacteria such as clostridium botulinum, listeria, and the like. Through retrieval, no literature report of adopting lactic acid bacteria and metabolites thereof to inhibit anthracnose pathogenic bacteria or prevent cucumber anthracnose exists at present.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide a compound lactobacillus preparation capable of preventing and treating cucumber anthracnose and application thereof.

In order to achieve the technical purpose, the inventor finally obtains the composite antagonistic bacterium consisting of the lactobacillus plantarum P-8(Lactobacillus plantarum P-8) and the Bifidobacterium lactis V9(Bifidobacterium lactis V9) through a large number of tests, screening and optimizing by combining the research experiences of various lactic acid bacteria for many years. After the compound bacterial liquid is sprayed to cucumber seedlings with anthracnose, the result shows that the death rate in the seedling stage is greatly reduced. Based on the research result, the invention provides the following technical scheme: a lactobacillus preparation for preventing and treating cucumber anthracnose contains active bacteria including Lactobacillus plantarum P-8(Lactobacillus plantarum P-8) and Bifidobacterium Lactis V9(Bifidobacterium Lactis V9).

The lactobacillus plantarum P-8(Lactobacillus plantarum P-8) adopted by the invention is separated from traditional natural fermented yoghurt of inner Mongolia, has excellent capability of resisting digestive juice of gastrointestinal tract, and can survive in animal intestinal tracts; the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of Xilu No.1 of Suo Xingyang district, Beijing, China, the preservation time is 2012, 06 and 28 days, and the preservation number of the strain is CGMCC No. 6312. The acid-resistant and bile salt-resistant probiotics separated from intestinal tracts of healthy Mongolian children in 2005 by the Bifidobacterium Lactis V9(Bifidobacterium Lactis V9) can inhibit harmful flora, protect intestinal mucosa barrier and metabolize to generate organic acid, and has high survival rate in the environment of pH 2.0-8.0. The strain is classified as Bifidobacterium animalis subsp.lactis, and has been preserved in China general microbiological culture Collection center (CGMCC No. 5470) at 11/18 days 2011.

Further preferably, the lactobacillus preparation for preventing and treating cucumber anthracnose as described above has a ratio of viable bacteria of lactobacillus plantarum P-8 to viable bacteria of bifidobacterium lactis V9 of 1: (3-8). Still further preferably, the ratio of viable bacteria of lactobacillus plantarum P-8 and bifidobacterium lactis V9 in the lactobacillus preparation is 1: (5-6). In a most preferred embodiment of the invention, the ratio of viable bacteria of lactobacillus plantarum P-8 and bifidobacterium lactis V9 in the lactic acid bacteria preparation is 1: 5.

it should be noted that the inventors of the present invention have unexpectedly found that Lactobacillus plantarum P-8 and Bifidobacterium lactis V are responsible for screening antagonistic bacteria against colletotrichum in cucumber by the plate confrontation method9 has certain antagonistic action on cucumber anthracnose, but the effect is not obvious; when the mixed fermentation liquor of the two bacteria and the anthracnose pathogen of cucumber are subjected to plate opposition, an extremely obvious antagonistic effect is shown. Further, the cucumber anthracnose pot inoculation test result shows that the mortality of sick seedlings is greatly reduced after the compound bacterium liquid of the lactobacillus plantarum P-8 and the bifidobacterium lactis V9 is sprayed on cucumber weak seedlings suffering from anthracnose. Therefore, the invention provides the application of the compound lactobacillus preparation in preventing and treating cucumber anthracnose. Further preferably, in the application of preventing and treating cucumber anthracnose, the lactobacillus preparation is prepared into a bacterial liquid and then sprayed on the leaf surfaces of cucumber seedlings, or/and the lactobacillus preparation is prepared into a bacterial liquid and then poured on the roots of the cucumber seedlings. Still further preferably, the viable bacteria concentration of the bacterial liquid is 2 x 10 when the bacterial liquid is sprayed⁶～8×10⁶CFU/ml; the viable bacteria concentration of the bacterial liquid is 2 multiplied by 10 when in pouring⁸～8×10⁸CFU/ml。

Compared with the prior art, the Lactobacillus preparation consists of Lactobacillus plantarum P-8(Lactobacillus plantarum P-8) and Bifidobacterium Lactis V9(Bifidobacterium Lactis V9), and the mortality of sick seedlings is greatly reduced after the Lactobacillus plantarum P-8 and the Bifidobacterium Lactis V9 are sprayed on cucumber seedlings with anthracnose as compound bacterial liquid, so that the cucumber anthracnose can be prevented and treated instead of chemical pesticides, the disease prevention potential of the birth microorganism prevention can be better played, a foundation is laid for finally planting and cultivating high-quality green cucumber food which is safe to human bodies, and the continuously improved consumption level of consumers can be better met.

Drawings

FIG. 1 shows the results of a cucumber anthracnose pot inoculation test;

Detailed Description

The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above aspects of the present invention are within the scope of the present invention. In addition, the experimental methods in the following examples are all conventional methods unless otherwise specified.

Example 1: preparation of the lactobacillus preparation:

preparation of MRS liquid culture medium:

the required materials are as follows:

preparing a liquid MRS culture medium: 1 liter of distilled water was dissolved the following components: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium phosphate, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 20 g of glucose, 801 ml of tween, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate and 1815 g of agar powder. Adjusting pH to 6.2-6.6, and autoclaving (101Kpa at 121 deg.C) for 15 min.

Preparing a skim milk culture medium: recovering skimmed milk powder to obtain skimmed milk at a ratio of 11%, naturally adjusting pH, and sterilizing at 115 deg.C for 20 min.

Activating strains and preparing fermented milk:

1. materials:

1)500ml of MRS liquid medium;

2)10 triangular bottles of 250ml, rubber plugs, kraft paper and ropes;

3)10 culture dishes;

4)1 inoculating loop, alcohol lamp, lighter and alcohol;

(1) respectively carrying out streak inoculation on 3-7% of inoculum size of the lactobacillus plantarum P-8 and the lactobacillus bifidus V9 in MRS liquid culture medium, culturing for 24-35 hours at 35 +/-1 ℃, centrifugally washing, adding 10-12% of sterilized and degreased milk on thalli precipitates, and adjusting the bacterial count to 1.9-2.3 multiplied by 10¹⁰cfu/mL, mixing uniformly, and then pouring and culturing;

(2) respectively inoculating the strain cultures obtained in the step (1) into MRS culture solution, culturing for 30-36 hours at 35 +/-1 ℃, then respectively inoculating 10-12% of sterilized degreased milk, culturing for 10-18 hours at 36 +/-1 ℃, and adjusting the number of viable bacteria to be 2.8-3.4 multiplied by 10¹⁰cfu/mL, pH 6.5-7.0.

Example 2: bacteriostasis experiment for determining cucumber anthracnose bacteria by using plate confronting method

Selecting a punch with the diameter of 0.6cm, and beating the activated cucumber anthracnose germs on the PDA flat plate into bacterial slices. And taking out qualified cucumber anthracnose bacterial slices, and transferring the cucumber anthracnose bacterial slices to the center of another PDA (personal digital assistant) plate. The activated various lactic acid bacteria on the PDA plate are beaten into bacterial slices by the same method, and are respectively and symmetrically inoculated at the positions 3cm away from the anthracnose pathogen, a blank Control (CK) is only inoculated with the anthracnose pathogen of cucumber, and the culture is carried out in the environment of 26 ℃. When the pathogenic bacteria of the blank control group overgrow the flat plate, the average colony diameter of the anthracnose of cucumber in each experimental group and the average bacteriostatic bandwidth of each lactic acid bacterium are measured, and the strength of the antagonistic action is measured according to the average bacteriostatic rate and the average bacteriostatic bandwidth of the lactic acid bacterium. The bacteriostatic bandwidth is the distance between the lactic acid colony and the edge of the pathogenic colony. The bacteriostasis rate is [ (CK pathogen colony diameter-experimental group pathogen colony diameter)/CK pathogen colony diameter ] × 100%.

In the test, 6 lactic acid bacteria, namely lactobacillus paracasei Probio-37(CGMCC No.18638), bifidobacterium lactis V9, lactobacillus gasseri G098 (1) L.pallidum, characteristics of antibacterial substances produced by enterococcus faecium BZ2 are studied [ D ] Nemond agricultural university, 2017 ]), lactobacillus plantarum IMAU94388 (1) Wang Shadong, high-density culture research of the enterococcus faecium IMAU94388 [ D ] Nemond agricultural university, 2020.), lactobacillus plantarum P-8 and lactobacillus rhamnosus R7970 (1) Liu industry, research of preparation technology of direct vat set type lactic acid bacteria leavening agent and application thereof [ D ] Shandong agricultural university, 2017 ]) are adopted, 10 culture medium flat plates are selected for each group to face each other, the average diameter of cucumber colonies is finally obtained, and the average antibacterial rate is calculated.

Table 1: antagonistic action of different strains on cucumber colletotrichum

Table 1 shows antagonistic test data of different strains on cucumber colletotrichum gloeosporioides, and it can be seen from the bacteriostatic rate that Bifidobacterium lactis V9 and Lactobacillus plantarum P-8 have obvious antagonistic action on cucumber colletotrichum gloeosporioides, the inhibition action is strongest, and the effect is most obvious. From the comparison of the initial bacteriostasis rates, the antibacterial rate is about 40% or more. As can be seen from the bacteriostatic bandwidth, Lactobacillus paracasei Probio-37 and Lactobacillus gasseri G098 also have certain antagonistic action, but the action is not obvious and is respectively 0.10cm and 0.30cm, and Bifidobacterium lactis V9 and Lactobacillus plantarum P-8 have obvious antagonistic action (the bacteriostatic bandwidth is more than or equal to 0.5cm) and are respectively 1.00cm and 0.70 cm.

Example 3: disease prevention test for antagonistic lactic acid bacteria

(1) Cutting stem tip of cucumber pod into segments with length of 1cm, soaking in distilled water containing 5% mercuric chloride for 30 s, and sterilizing. Then inoculating the cucumber anthracnose mycelium blocks to the sterilized vegetable pods by using an inoculating needle on a superclean workbench, sealing the ports, putting the pods on a culture rack, culturing for 7 hours at a constant temperature of 24 ℃, pouring 20-25ml of sterile water, and uniformly stirring.

(2) The suspension of lactic acid bacteria (prepared in example 1) was diluted with sterile water and shaken up in a shaker to control the number of colonies in the bacterial suspension to 3X 10⁶The volume of the solution is kept for later use. When new leaves grow on cucumber seedlings, the prepared lactobacillus plantarum P-8, bifidobacterium lactis V9 and lactobacillus suspension and sterile water are respectively used for intervention treatment.

(3) In this example, a blank control group (sterile water), a lactobacillus plantarum P-8 group, a bifidobacterium lactis V9 group, a complex bacteria a group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:1), a complex bacteria B group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:3), a complex bacteria C group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:5), and a complex bacteria D group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:9) were provided, 50 test seedlings were selected for each group, and an arc-shaped booth with a radius of 50 cm was constructed with a plastic film to ensure that the temperature in the booth was between 20 and 25 ℃, the relative humidity of the soil was 85% to 90%, the relative humidity of the air was 70% to 90%, and the temperature of the booth was too high, and the booth was ventilated to lower the humidity. Spraying for 1 time every 9 am, spraying with an electric small spraying pot, adjusting the spraying state with a sprayer, spraying from top to bottom at an angle of 45 degrees from the oblique upper side of the seedling to ensure that the front and back sides of the leaf are sprayed, spraying 5ml each (spraying 2.5ml each of lactobacillus plantarum P-8 and bifidobacterium lactis V9 on a compound bacterium group A, spraying 1.25ml and 3.75ml respectively on lactobacillus plantarum P-8 and bifidobacterium lactis V9 on a compound bacterium group B, spraying 0.8ml and 4.2ml respectively on lactobacillus plantarum P-8 and bifidobacterium lactis V9 on a compound bacterium group C, spraying 0.5ml and 4.5ml respectively on lactobacillus plantarum P-8 and bifidobacterium lactis V9 on a compound bacterium group D), spraying for 3 days, spraying for 1 time every day, and counting the disease incidence level and disease index at 17 pm on the third day. The calculation formula of the disease index is sigma, the disease number of each level is multiplied by the corresponding severity/survey total number is multiplied by 100 percent

Table 2: cucumber seedling morbidity level judgment standard

Grade of disease onset	Severity degree
		Level 0	No scab
Level 1	The inoculation point has slight small spot with diameter less than 0.5cm
		Stage 2	The disease spots are obvious, and the diameter is 0.5-1.0cm
Grade 3	The lesion spot has a diameter of 1cm and occupies a leaf area of 1/3
		4 stage	The lesion area accounts for 1/3-2/3
Grade 5	The area of the lesion spots is more than 2/3

Table 3: the disease level and disease index of each group are compared

As shown in the comparative test data of the disease grade numbers and the disease indexes of each group in the table 3, the control effect sequence of the anthracnose of the cucumber is as follows from big to small: the conclusion can be drawn when the ratio of the composite bacteria group C to the composite bacteria group B to the bifidobacterium lactis group V9 to the composite bacteria group A to the lactobacillus plantarum group P-8 to the composite bacteria group D to the blank control group is as follows: when the ratio of the bifidobacterium lactis V9 to the bifidobacterium lactis V9 is 1:5, the control effect on cucumber anthracnose is best.

Example 4: cucumber anthracnose pot inoculation test

In this example, a blank control group (sterile water), a lactobacillus plantarum P-8 group, a bifidobacterium lactis V9 group, a composite bacteria a group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:1), a composite bacteria B group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:3), a composite bacteria C group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:5), and a composite bacteria D group (lactobacillus plantarum P-8: bifidobacterium lactis V9 ═ 1:9) were set, and 350 anthrax test seedlings were randomly selected from each group. The test seedlings are separated by 15 cm per pot, and the test seedlings in each group are separated by a plastic film with the height of 1.2 m, so that the cross infection between the spraying preparation and the agricultural operation is prevented, the temperature in the greenhouse is ensured to be between 18 and 28 ℃, the relative humidity of soil is between 70 and 90 percent, the relative humidity of air is between 60 and 90 percent, and the high morning temperature can ventilate, cool and reduce the dampness of the greenhouse film.

The suspension of lactic acid bacteria (prepared in example 1) was diluted with sterile water and shaken up in a shaker to control the number of colonies in the bacterial suspension to 3X 10⁶Per ml preparationThe application is as follows. Measuring a lactic acid bacteria liquid by using a measuring cylinder, pouring into a basin, directly root-pouring 100 times of diluent, pouring 250ml of each seedling (125 ml of each of a composite bacteria group A root-poured lactobacillus plantarum P-8 and bifidobacterium lactis V9, 62.5ml and 187.5ml of each of a composite bacteria group B root-poured lactobacillus plantarum P-8 and bifidobacterium lactis V9, 40ml and 210ml of each of a composite bacteria group C root-poured lactobacillus plantarum P-8 and bifidobacterium lactis V9, 25ml and 225ml of each of a composite bacteria group D root-poured lactobacillus plantarum P-8 and bifidobacterium lactis V9), pouring once every five days, and counting the death rate of the seedlings at 16 noon on the same day, wherein the test period is 15 days, 3 times of pouring are carried out, and 3 times of death rate are counted. The death rate is 1-sigma, the survival number of each stage of statistical test/the number of the tested strains/the statistical number is multiplied by 100%.

Table 4: comparison of survival and death rate of each group during the test period

As can be seen from each group of data in Table 4, the two strains selected by the invention have certain control effect on cucumber anthracnose, and the death rate is from small to orderly composite bacteria C group < composite bacteria B group < composite bacteria A group < Bifidobacterium lactis V9 group < composite bacteria D group < Lactobacillus plantarum P-8 group < blank control group. The composite bacteria group C has the best effect (figure 1), the control effect on cucumber anthracnose is 90.67%, and the seedling death rate is 9.33%. The prevention and treatment effect of the bifidobacterium lactis V9 on cucumber anthracnose is 89.81%, and the seedling death rate is 10.19%. The control effect of the compound bacterium B group on cucumber anthracnose is 89.81%, and the death rate in seedling stage is 10.19%. The control effect of the compound bacterium A group on cucumber anthracnose is 89.71%, and the death rate in seedling stage is 10.29%. The control effect of the compound bacterium D group on cucumber anthracnose is 79.63%, and the death rate in seedling stage is 21.33%. The control effect of the lactobacillus plantarum P-8 group is 77.33%, and the death rate in the seedling stage is 22.67%. The prevention and treatment effect of the sterile water group is poor, the prevention and treatment effect is 68%, and the death rate in the seedling stage is 32.00%. The mortality rate of each treated seedling is significantly different from that of the sterile water group.

In the prior reports, antagonism is generally summarized as an effective substance formed from a protein compound or an organic acid. Through intensive research, the inventor discovers the inhibition effect on cucumber anthracnose bacteria in the process of determining the inhibition rate of lactic acid bacteria, and the inhibition spectrum is wider, which indicates that the lactic acid bacteria have a certain antibacterial effect on the cucumber anthracnose bacteria. The inhibition is to obtain organic acid by fermentation, thereby better inhibiting fungi, and inhibiting pathogen and fungi transmission. According to the dynamic research of the cucumber leaves, the metabolic products of the lactic acid bacteria have good control effect on the cucumber leaves. The research of the invention provides a foundation for effectively controlling the anthracnose of the cucumber.

Claims

1. A lactobacillus preparation for preventing and treating cucumber anthracnose is characterized in that active bacteria in the lactobacillus preparation consist of lactobacillus plantarum P-8 and bifidobacterium lactis V9.

2. The lactobacillus preparation for preventing and treating cucumber anthracnose according to claim 1, wherein the ratio of viable bacteria of lactobacillus plantarum P-8 to viable bacteria of bifidobacterium lactis V9 in the lactobacillus preparation is 1: (3-8).

3. The lactobacillus preparation for preventing and treating cucumber anthracnose according to claim 2, wherein the ratio of viable bacteria of lactobacillus plantarum P-8 to viable bacteria of bifidobacterium lactis V9 in the lactobacillus preparation is 1: (5-6).

4. Use of the lactic acid bacteria preparation according to claim 1, 2 or 3 for the control of cucumber anthracnose.

5. The application of the lactobacillus preparation in preventing and treating cucumber anthracnose according to claim 4, wherein the lactobacillus preparation is prepared into a bacterial solution and then sprayed on the leaf surfaces of cucumber seedlings, or/and the lactobacillus preparation is prepared into a bacterial solution and then poured on the roots of the cucumber seedlings.

6. The use of the lactic acid bacteria preparation according to claim 5 for the prevention and treatment of cucumber anthracnose, wherein the viable bacteria concentration of the bacterial liquid is 2 x 10 when spraying⁶～8×10⁶CFU/ml。

7. The use of the lactic acid bacteria preparation according to claim 5 for the prevention and treatment of cucumber anthracnose, wherein the viable bacteria concentration of the bacterial liquid is 2 x 10⁸～8×10⁸CFU/ml。