CN110387340B

CN110387340B - Lactobacillus plantarum L16 and application thereof in preventing and treating vegetable diseases

Info

Publication number: CN110387340B
Application number: CN201910608319.5A
Authority: CN
Inventors: 马丽; 曹立湘; 赵述淼; 赵星; 曹伟
Original assignee: Hubei Xinbaode Bio Tech Co ltd
Current assignee: Hubei Xinbaode Bio Tech Co ltd
Priority date: 2019-07-08
Filing date: 2019-07-08
Publication date: 2022-02-01
Anticipated expiration: 2039-07-08
Also published as: CN110387340A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum L16 and application thereof in preventing and treating vegetable diseases, wherein the lactobacillus plantarum disclosed by the invention is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and M2019502. The strain has good antagonistic action on the cucumber specialization types of colletotrichum, alternaria solani and fusarium oxysporum, and the inhibition rates of the three pathogenic bacteria are 44.29%, 40% and 45.71% respectively by a plate confronting method. The strain also has the stress resistance suitable for commercialization, and the survival rate of the strain after spray drying for three months is still more than 30 percent; meanwhile, the method also provides a process that the lactobacillus plantarum L16 is applied in combination with an organic fertilizer in the biological control of cucumber anthracnose in field planting of the strain, which can effectively reduce the morbidity by 30% and improve the production benefit.

Description

Lactobacillus plantarum L16 and application thereof in preventing and treating vegetable diseases

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum L16 and application thereof in preventing and treating vegetable diseases.

Background

Vegetables are main crops in agricultural production and are also essential dishes on the dining table of common people. In order to supply sufficient fresh vegetables to meet the needs of national life all the year round, greenhouse planting and continuous cropping are the most important vegetable production mode nowadays. But the greenhouse environment has poor ventilation, high temperature, weak sunlight intensity and high humidity, and increases the incidence rate of plant diseases and insect pests; the long-term continuous cropping and fertilizer application cause the destruction of the soil structure and also promote the increase of the disease incidence. Common diseases in vegetable cultivation, such as blight, root knot nematode, black spot and the like, can reduce the quality of vegetables, even cause large-area vegetable death, seriously reduce the production benefit and cause huge economic loss.

The green prevention and control technology of vegetable diseases and insect pests is an important means for continuously controlling the vegetable diseases and insect pests and guaranteeing the production safety of vegetables, and is an effective way for improving the quality safety of the vegetables and protecting the ecological environment. The green prevention and control technologies comprise good land management such as field cleaning, scientific fertilization, crop rotation and stubble rotation and the like, and effective biological prevention and control methods such as antibiotics, various plant extracts and biological agents and the like. The beneficial microorganisms are added into the soil by applying bacterial manure or biological agents, the added beneficial microorganisms can compete with pathogenic bacteria to change the soil microbial community structure, so that the soil microbial community structure is developed in a better direction, and meanwhile, the biocontrol agent can decompose soil organic matters, improve the nutritional conditions of crops and promote the growth of the crops. Commonly used biocontrol microbial agents include bacillus, mould, pseudomonas and the like.

Lactic acid bacteria are a group of bacteria that ferment carbohydrates to produce lactic acid and are widely distributed in nature. Lactic acid bacteria fermentation is a traditional food preservation technology, and the lactobacillin and organic acid generated by the metabolism of lactic acid bacteria can effectively inhibit the growth of bacteria and fungi, so that the storage period of food is prolonged. The antagonism of lactic acid bacteria to bacteria and fungi makes it have the potential as the plant biocontrol microbial inoculum, but the application research report on lactic acid bacteria in biocontrol at home and abroad is less at present, naked oats, etc. are screened from preserved beancurd and pickled vegetables to 3 strains of lactic acid bacteria with antagonism effect to the anthracnose germ plate of cucumber, wherein 1 strain of lactic acid bacteria has the disease incidence rate reduction more than 50% in the small-range experiment of potting and greenhouse; the spinach seed is treated for 24h by the lactobacillus pesticide developed by Japanese scientists, so that the incidence rate of spinach in spinach blight disease soil is reduced to 12 percent; the prevention and treatment effects of lactobacillus fermentation liquor separated from pomegranate such as Gajbhiye on grape downy mildew, anthracnose and white rot are respectively 91.8%, 2.9% and 95.1%, which are all superior to those of conventional chemical agents. The lactobacillus has various types and rich genetic diversity, and effective strains in the lactobacillus can be screened to be used as the vegetable biocontrol bacteria to antagonize crop pathogenic bacteria, so that the vegetable morbidity is reduced, and the quality and the yield are improved.

Disclosure of Invention

The invention aims to provide a lactobacillus plantarum with a biocontrol effect, wherein the lactobacillus plantarum is lactobacillus plantarum (L16) with a preservation number of CCTCC NO: and M2019502.

Another object of the present invention is to provide the use of Lactobacillus plantarum L16 as a biocontrol strain; the lactobacillus plantarum provided by the invention has a good inhibition effect on the special cucumber types of colletotrichum, alternaria solani and fusarium oxysporum, so that the lactobacillus plantarum has a good application prospect in prevention and treatment of vegetable diseases.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant separates lactobacillus from pickle, orchard rhizosphere soil, rotten leaves of vegetable field and chopped pepper samples, and further screens strains with the best specialized inhibition effect on colletotrichum, alternaria solani and fusarium oxysporum cucumber, so as to finally obtain a lactobacillus plantarum strain which is sent to the China center for type culture collection for collection in 28 days 6.2019, and is classified and named: lactobacillus plantarum L16, accession number: CCTCC NO: m2019502, address: wuhan university in Wuhan, China.

The lactobacillus plantarum L16 is a gram-positive coccus without spores. The colony on the MRS solid culture medium is milky white, the surface is smooth and moist, the colony is in a drop shape, a round shape, a bulge and a neat edge, and the diameter of the colony is about 3 mm. The strain is simply dyed and observed under a microscope, the shape of the strain is straight or bent rod-shaped, single, paired or chain-shaped, the size of the strain is 0.9-1.0um multiplied by 4.5-6.0 mu m, and the strain has no flagellum. Facultative anaerobic, no reduction of nitrate, no liquefaction of gelatin, negative for both catalase and oxidase. Can grow in gluconate and produce CO₂(ii) a Producing L-lactic acid by using glucose homolactic fermentation; fermentation of 1 molecule of ribose or other pentose sugar produces 1 molecule of lactic acid and 1 molecule of acetic acid. The optimum growth temperature is 35 ℃.

The application of lactobacillus plantarum L16 as a bio-control strain comprises that the lactobacillus plantarum is independently used as a bio-control microbial inoculum or is prepared into the bio-control microbial inoculum together with an organic fertilizer;

the biocontrol strain is used for preventing and controlling the cucumber specialization types of colletotrichum, alternaria solani or fusarium oxysporum.

Compared with the prior art, the invention has the following characteristics:

1) the invention provides a lactobacillus plantarum strain for the first time, which has the biocontrol characteristics of simultaneously antagonizing colletotrichum, alternaria solani, fusarium oxysporum (cucumber specialization type) and the like, so that the lactobacillus plantarum strain has a better application prospect in the prevention and treatment of vegetable diseases. The inhibition rates of the lactobacillus plantarum L16 on the three pathogenic bacteria measured by a plate confrontation method are 44.29%, 40% and 45.71% respectively.

2) The lactobacillus plantarum provided by the invention has good stress resistance, the number of viable bacteria after fermentation can reach 200 plus 260 hundred million CFU/ml, and the survival rate after three months of drying is still more than 30%.

3) The lactobacillus plantarum provided by the invention is sprayed on an organic fertilizer, and is applied to crops in a combined way, so that the lactobacillus plantarum has better control effect than that of the single application of bacterial liquid.

Drawings

FIG. 1 is a colony morphology of strain L16.

FIG. 2 is a simple staining microscopic morphology of strain L16.

FIG. 3 schematic diagram of the cultivation of Lactobacillus plantarum L16 in a 5L fermenter.

Fig. 4 is a schematic diagram showing the change of the number of living bacteria stored in the organic fertilizer containing lactobacillus plantarum L16.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

screening of Lactobacillus plantarum L16 Strain:

1. preparation of a culture medium:

BCP culture medium peptone 5 g; 3g of yeast extract; 5g of lactose; 20g of agar; 10ml of 0.5 percent bromocresol purple; 1000ml of distilled water; the pH value is 6.8-7.0.

MRS liquid medium: 20g/L of glucose; peptone 10 g/L; 5g/L of yeast extract; 10g/L of beef extract powder; 2g/L of diammonium citrate; 5g/L of sodium acetate; tween 801 g/L; k2HPO 42 g/L; MgSO4 & 7H2 O0.058g/L; MnSO 4.4H 2O 0.25.25 g/L; 1000ml of distilled water; pH6.5-6.8.

PDA culture medium: 200g of potatoes; 20g of glucose; 15-20 g of agar; 1000ml of distilled water; naturally P H.

Solid medium 1.5% agar was added based on liquid medium and all media were sterilized at 115 ℃ for 20 min.

2. Primary screening: taking 5g of pickle, orchard rhizosphere soil, vegetable field rotten leaves and chopped pepper samples, placing the pickle, orchard rhizosphere soil, vegetable field rotten leaves and chopped pepper samples in 300ml of MRS liquid culture medium for culturing for 12h, taking 1ml of culture solution for gradient dilution, coating the culture solution on a BCP plate, culturing at 37 ℃ for 48h, selecting bacterial colonies with typical lactic acid bacteria colony morphology (bacterial colony periphery is yellow) for streak separation culture, selecting about 5 bacterial colonies for each sample, transferring the obtained single bacterial colonies to the MRS liquid culture medium for culturing for 12h, and performing MRS slant preservation.

3. Re-screening: performing plate secondary screening on the 20 strains (numbered L1-L20) obtained by primary screening, and respectively detecting the bacteriostatic properties of the strains aiming at three common plant pathogenic bacteria of tomato early blight (pathogenic bacteria: alternaria solani), cucumber anthracnose (pathogenic bacteria: colletotrichum gloeosporioides) and cucumber fusarium wilt (fusarium oxysporum cucumber specialization type), wherein the specific steps are as follows:

using a puncher with the diameter of 0.6cm to punch a cultured pathogenic bacterium strain sheet on a PDA plate, transferring the sheet to the center of another PDA plate, using a pick needle to symmetrically inoculate activated lactic acid bacteria (with the number of L1-L20) at a position 3cm away from pathogenic bacteria (3 cm away from the colony edge of the pathogenic bacteria at the center of the plate), inoculating only the pathogenic bacteria in a contrast way, and then culturing at the constant temperature of 26 ℃. When the contrast is full of the flat plate, the average colony diameter of pathogenic bacteria and the average bacteriostatic bandwidth of the lactic acid bacteria are measured, the bacteriostatic rate is calculated, and the strength of the antagonistic action is measured according to the average bacteriostatic rate and the average bacteriostatic bandwidth of the lactic acid bacteria.

Finally, the strain L16 is screened out to show the best antagonistic effect on three pathogenic bacteria, the plate inhibition rate on colletotrichum is 44.29%, the inhibition rate on alternaria solani is 40%, and the inhibition rate on fusarium oxysporum cucumber transformation is 45.71% (Table 1). The strain L16 shows better biocontrol potential, and then the strain L16 is taken as a target for subsequent research.

TABLE 1

Biochemical identification of strain L16: the selected objective strain L16 was briefly stained with crystal violet and observed by an optical microscope. The strain was found to be a gram-positive coccus, without spores, in the shape of straight or bent rods, single, paired or chained (fig. 2). The colony on MRS solid medium is milky white, has smooth and moist surface, drop shape, circular shape, raised shape and regular edge, and the diameter of the colony is about 3mm (figure 1). The physiological and biochemical identification strain L16 is facultative anaerobic, does not reduce nitrate, does not liquefy gelatin, and is negative to both catalase and oxidase. Can grow in gluconate and produce CO₂(ii) a Producing L-lactic acid by using glucose homolactic fermentation; fermentation of 1 molecule of ribose or other pentose sugar produces 1 molecule of lactic acid and 1 molecule of acetic acid. The optimum growth temperature is 35 ℃.

16s identification of the strains: and (3) selecting an L16 single colony, amplifying the single colony by using a universal primer, carrying out 16S LLNA sequencing analysis on an amplification product and constructing a phylogenetic tree. The result shows that the sequence has the highest similarity with 16S LDNA sequences of Lactobacillus plantarum subsp.aLgeno Latens and Lactobacillus plantarum subsp.plantarum, and the similarity reaches 99 percent, so that the strain is determined to be Lactobacillus plantarum and is named as Lactobacillus plantarum L16. The strain is delivered to China center for type culture Collection in 2019, 6 and 28 days, and is classified and named as follows: lactobacillus plantarum L16, accession number: CCTCC NO: m2019502, address: wuhan university in Wuhan, China.

Example 2:

high-density culture of lactobacillus plantarum L16:

fermentation medium: 50g/L of glucose; 15g/L of corn starch; 5g/L of yeast extract; peptone 5 g/L; 2g/L of diammonium citrate; 5g/L of sodium acetate; tween 801 g/L；K₂HPO₄ 2g/L；MgSO₄·7H₂O 1.0g/L；M nSO₄·H₂O 0.25g/L；FeSO₄·7H₂O0.5 g/L; sterilizing at 115 deg.C for 20min at pH of 6.5-6.8.

A supplemented medium: glucose 200g/L, and autoclaving at 115 deg.C for 20 min.

The experiment was carried out using a 5L fermentor. 2.5L of fermentation medium is prepared according to the formula, the initial pH is adjusted to 6.5, the temperature is controlled to be 35 ℃ in the fermentation process, and the mixture is stirred at 150 rpm. Inoculating lactobacillus plantarum L16 seed liquid according to the inoculation amount of 5 percent to start fermentation, adjusting the pH value by using a 20 percent NaOH solution, and controlling the constant pH value to be 6.0. After 24h of fermentation, continuous feeding (feeding speed 3.00ml/min) was carried out with a feeding volume of 500 ml. Reducing sugar concentration, OD600 and pH were measured at every 6h sampling during the fermentation (FIG. 3).

The results show that the pH value is always kept about 6.0 in the fermentation process, the sugar is completely consumed in 48 hours, and the total consumption of glucose is 80g/L after the fermentation is finished. The final OD600 can reach 30, and the viable count measured by a dilution plate method reaches 267 multiplied by 10⁸CFU/ml。

300L pilot fermentation: the lactobacillus plantarum L16 strain is activated by MRS liquid culture medium, transferred into 300ml of liquid culture medium, inoculated with 1 percent of inoculum concentration, and kept stand for 24 hours at 35 ℃. Sterilizing 300L fermentation medium, cooling to 35 deg.C, inoculating the seed solution, stirring at 100Lpm for 35 deg.C, adjusting with 20% NaOH solution for pH 6.0 + -0.2, fermenting for 24 hr, directly adding 10kg glucose powder, fermenting for 48 hr, and discharging. The final viable count was 255X 10 by dilution plate method⁸CFU/m l。

The bacterial liquid is subjected to spray drying by using a conventional method in the field and then is stored at a constant temperature of 20 ℃, and the survival rate of the bacterial liquid is still more than 30% after the bacterial liquid is dried for three months.

Example 3:

processing and preservation of lactobacillus plantarum L16:

the fermentation liquor tested in example 2 was concentrated to 1/3 by centrifugation at 3000rpm, and 1% sodium carboxymethylcellulose and 1% sodium alginate were added and homogenized, and then sprayed onto granulated organic fertilizer granules, 20kg per ton of organic fertilizer. Packaging into a sealed bag, storing at constant temperature of 20 deg.C, sampling every other week to detect viable count, and continuously monitoring for 3 months.

As can be seen from FIG. 4, the number of viable bacteria was 5X 10 after 1 month storage⁸CFU/g, survival rate is more than 30%; the number of viable bacteria is still 3.9 × 10 after three months of storage⁸CFU/g, survival rate 26%. The biological organic fertilizer containing the lactobacillus plantarum L16 shows that a large amount of bacteria can be inactivated when stored for three months, but partial viable bacteria are remained.

Example 4:

the application of the lactobacillus plantarum L16 in preventing and treating vegetable diseases comprises the following steps:

pot experiment: and (3) carrying out seedling raising of cucumber seeds in a seedling tray, transplanting seedlings with consistent growth vigor to a flowerpot after the seedlings grow into 2 leaves, wherein the number of the seedlings in the flowerpot is 60, and each pot contains 3 plants. The pot culture soil is mixed with the fusarium oxysporum with the addition amount of 10^⁴CFU/g (dry soil). Dividing 60 plants into three treatment groups and control group at random, respectively using 0.1ml, 1ml and 10ml Lactobacillus plantarum L16 bacterial liquid (effective bacterial concentration of bacterial liquid is 5 × 10)⁹CFU/ml) to 10ml, and the control group was irrigated with 10ml water once a week for a test period of 28 days. And (5) counting the morbidity and disease index after the test is finished.

TABLE 2 prevention and treatment of wilt of potted cucumber with Lactobacillus plantarum L16

Lactobacillus plantarum L16 was used for antagonistic experiments against a potted plant of colletotrichum (cucumber anthracnose) and Alternaria solani (tomato early blight) by methods such as Fusarium oxysporum. The results are shown in tables 3 and 4.

TABLE 3 prevention and control of anthracnose of potted cucumber by Lactobacillus plantarum L16

TABLE 4 prevention and treatment effects of Lactobacillus plantarum L16 on early blight of potted tomato

From tables 2-4, it can be seen that lactobacillus plantarum L16 has better control effects on cucumber fusarium wilt, cucumber anthracnose and tomato early blight, the control effect is better when the application amount is larger, the disease incidence can be reduced by about 50%, and the control effects on the three diseases are respectively 62.7%, 51.8% and 43.2%.

Field test: placing cucumber seeds in a seedling tray for seedling culture, growing into 2-leaf seedlings, transplanting the seedlings with consistent growth vigor into a disease field with cucumber anthracnose outbreak, and dividing into 3 treatment groups and a control group, wherein 6000 plants are total. The treatment group 1 uses organic fertilizer containing lactobacillus plantarum L16, the treatment group 2 uses organic fertilizer without lactobacillus plantarum L16, and the treatment group 3 uses lactobacillus plantarum L16 fermentation liquor (the effective bacteria concentration of the bacteria liquid is 5 multiplied by 10)⁹CFU/ml)10 ml/plant, the

treatment groups

1 and 2 and the control group were filled with 10 ml/plant of clear water once a week for 35 days of the test period. 20kg of lactobacillus plantarum L16 fermentation liquor is sprayed on each ton of organic fertilizer applied by the treatment group 1, (after being sealed by the organic fertilizer sprayed with lactobacillus plantarum L16, 34.6% of live bacteria still exist after being stored for 30 days at 20 ℃), and 50g of organic fertilizer base fertilizer is applied on each cucumber seedling. And (5) counting the morbidity after the test is finished.

TABLE 5 prevention and treatment effect of Lactobacillus plantarum L16 on anthracnose of cucumber in field

The result shows that the incidence rate of cucumber anthracnose of the cucumber in the continuous cropping cucumber in the diseased field can reach 54.54 percent if the cucumber is not treated, and the incidence rate can be obviously reduced by root irrigation with the lactobacillus plantarum L16 bacterial liquid, so that the incidence rate of the cucumber anthracnose is reduced to 15.46 percent. The morbidity can be reduced by about 30% by applying the organic fertilizer, and the morbidity can be reduced to 10.8% by using the organic fertilizer and the lactobacillus plantarum L16 together. The lactobacillus plantarum L16 in a field experiment has an ideal biocontrol effect on cucumber anthracnose, can be used as an ideal control means in production, and has a better biocontrol effect when being combined with organic fertilizers.

Claims

1. Separated plant milkThe lactobacillus plantarum is lactobacillus plantarum (A)lactobacillus plantarum) L16 with a preservation number of CCTCC NO: and M2019502.

2. Use of the lactobacillus plantarum of claim 1 in the preparation of a biocontrol microbial inoculum.

3. Use of the lactobacillus plantarum described in claim 1 for the preparation of a colletotrichum bacteriostat.

4. Use of the lactobacillus plantarum of claim 1 for the preparation of a bacterial inhibitor of alternaria solani.

5. Use of the lactobacillus plantarum described in claim 1 for the preparation of a fusarium oxysporum cucumber-specific bacteriostatic agent.

6. The use of lactobacillus plantarum as defined in claim 1 in the preparation of a bio-control fertilizer in combination with an organic fertilizer.

7. The use of claim 6, said biocontrol fertilizer is used for controlling cucumber anthracnose.