阅读说明：本技术 一种用于抑制采后果实腐败病原菌的含铁离子菌悬液及其制备方法和应用 (Iron ion-containing bacterial suspension for inhibiting postharvest fruit putrefaction pathogenic bacteria and preparation method and application thereof ) 是由 周文文 叶婉琼 聂麟杰 于 2021-07-12 设计创作，主要内容包括：本发明公开了一种用于抑制采后果实腐败病原菌的含铁离子菌悬液及其制备方法和应用。通过在一定培养阶段的芽孢杆菌菌悬液中添加金属铁离子提高芽孢杆菌生物膜的形成及抑制果实腐败菌的能力。在体外,含有金属化合物的菌悬液对水果腐败菌(扩展青霉、灰葡萄孢霉)的抑制效果显著增强；在体内,对樱桃番茄、枇杷、梨贮藏中腐败菌(扩展青霉、灰葡萄孢霉)的抑制能力明显提升。本发明建立生防菌株解淀粉芽孢杆菌B4(Bacillus amyloliquefaciens B4)生物被膜形成与其在生防作用中的联系,外源添加金属化合物显著提高解淀粉芽孢杆菌B4生物被膜形成能力和对青霉、灰霉的体内及体外抑制作用；操作简单,易于实用,有助于解淀粉芽孢杆菌产品化。(The invention discloses an iron ion-containing bacterial suspension for inhibiting postharvest fruit putrefactive pathogenic bacteria, and a preparation method and application thereof. The metal iron ions are added into the bacillus bacterial suspension in a certain culture stage to improve the formation of a bacillus biofilm and the capability of inhibiting fruit putrefying bacteria. In vitro, the inhibition effect of the bacterial suspension containing the metal compound on fruit putrefying bacteria (penicillium expansum and botrytis cinerea) is obviously enhanced; in vivo, the inhibition ability of putrefying bacteria (penicillium expansum and botrytis cinerea) in storage of cherry tomatoes, loquats and pears is obviously improved. The invention establishes the connection between the formation of a biofilm of a biocontrol strain Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) and the biocontrol effect thereof, and the exogenous addition of a metal compound obviously improves the biofilm formation capability of the Bacillus amyloliquefaciens B4 and the in-vivo and in-vitro inhibition effect on penicillium and botrytis cinerea; simple operation, easy practicality and contribution to the production of the bacillus amyloliquefaciens.)

1. A preparation method of a suspension containing iron ions for inhibiting postharvest fruit putrefactive pathogenic bacteria is characterized by comprising the following steps:

(1) activating a Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) strain on a plate culture medium, selecting a single colony, inoculating the single colony into a test tube containing an LB liquid culture medium, and culturing at 37 ℃, 180 and 220rpm for 12-16h to obtain a Bacillus amyloliquefaciens seed culture solution;

the Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13466 and the preservation date of 2016, 12 and 19 days;

(2) inoculating the seed culture solution into LB liquid culture medium according to the volume ratio of 1.0-2.0%, culturing at 37 deg.C for 4-6h, and centrifuging the fermentation liquid at 20-30 deg.C and 5000-10000g for 2-6min to obtain thallus.

(3) And (3) re-suspending the thallus obtained in the step (2) by using an LB liquid culture medium containing iron ions to obtain an iron ion-containing bacteria suspension.

2. The method according to claim 1, wherein in the step (3), LB liquid medium containing iron ions is used as a basisSuspending to the concentration of 1X 10 bacterial suspension B4⁴-1×10⁷CFU/mL。

3. The method according to claim 1 or 2, wherein the concentration of the iron ion in the bacterial suspension is 0.3 to 1.0 mM.

4. A suspension containing ferric ions for inhibiting postharvest fruit spoilage pathogens produced by the method of any one of claims 1 to 3.

5. Use of the iron ion-containing bacterial suspension according to claim 5 for inhibiting post-harvest fruit spoilage pathogens, wherein the iron ions are present in the form of ferric chloride, ferric sulfate, ferric nitrate.

6. Use of the iron ion-containing bacterial suspension according to claim 4 for inhibiting postharvest fruit spoilage pathogens, wherein the fruit is pear, loquat, cherry tomato.

7. Use according to claim 6, characterized in that: the postharvest fruit spoilage pathogenic bacteria comprise: penicillium expansum, Botrytis cinerea.

8. Use according to claim 6, characterized in that: the method is applied to biological control of postharvest fruit spoilage pathogens in any one of the following modes:

mode A: the concentration is 1 x 10⁴-1×10⁷Spraying the CFU/mL suspension containing the iron ions on the surfaces of the picked fruits to wet the surfaces of the fruits; after treatment, standing and airing at room temperature and under the condition that the relative humidity is 40% -90%;

mode B: soaking picked fruit in 1 × 10⁴-1×10⁷Soaking in CFU/mL iron ion-containing bacteria suspension for 0.5-2.5 min; after treatment, standing and airing at room temperature and under the relative humidity of 40% -90%.

Technical Field

The invention relates to the technical field of biological control bacteria on postharvest diseases of fruits, in particular to an iron ion-containing bacteria suspension for inhibiting postharvest fruit putrefactive pathogenic bacteria, and a preparation method and application thereof.

Background

Biofilms are structural bacterial communities attached to inert or living surfaces, where the bacteria are encapsulated in a network of hydrated extracellular polymeric matrices. Extracellular polymers are composed primarily of polysaccharides, proteins, nucleic acids and lipids, which provide mechanical stability to the biofilm, interconnecting and temporarily immobilizing bacterial cells. The exchange of nutrients in the extracellular polymeric substances allows the bacteria to form biofilms of considerable thickness while still maintaining the individual cells in an optimal nutritional state.

Bacillus subtilis, a non-pathogenic gram-positive bacterium, has become a model organism for studying biofilm formation. The bacillus subtilis has a complex regulation network and can respond to constantly changing environmental conditions to coordinate the expression of genes related to the biofilm matrix. Research shows that the stronger the biofilm formation capability of the bacillus subtilis is, the more remarkable the plant disease control effect is. Therefore, the research on the forming capability and forming conditions of the biological membrane is beneficial to the genetic improvement research and reasonable use of the biocontrol bacteria in the future.

Bacillus amyloliquefaciens is a gram-positive bacterium, has high affinity with Bacillus subtilis, and is widely distributed in natural environment. The bacillus amyloliquefaciens is considered to be an ideal biocontrol bacterium resource due to the broad-spectrum activity of inhibiting fungi and bacteria, high stability, strong stress resistance and high growth speed. The bacillus amyloliquefaciens B4 can form an extremely obvious biofilm under the standing condition, and 308 genes are annotated to the molecular binding in the first-class molecular function category, so that the biofilm formation is improved by adding harmless metal seeds from an external source on the basis of the existing biocontrol capability, the improvement of the capability of inhibiting putrefactive pathogenic bacteria is realized, and a new idea is provided for biological control of postharvest fruit diseases.

1.Costerton J W,Stewart P S,Greenberg E P.Bacterial biofilms:a common cause of persistent infections[J].Science,1999,284(5418):1318-1322.

2. Functional research of bacillus subtilis biomembrane in bacterial wilt biocontrol and Cyclic-di-GMP signal pathway preliminary exploration [ D ]. Nanjing agriculture university, 2012.

Disclosure of Invention

The invention aims to provide a method for improving the capability of biocontrol bacteria in inhibiting putrefying bacteria by adding metal ions from an external source and an application method in fruit storage. According to the method, on the premise of no external source harm, the metal iron ions are added into the bacterial suspension, so that the formation of a bacillus biofilm is enhanced, the capability of bacillus in inhibiting pathogenic bacteria in a culture plate and fruits is improved, the morbidity of the picked fruits is reduced, and a foundation is laid for a new method for enhancing the biocontrol effect of strains.

The invention is realized by the following technical scheme:

the invention firstly provides a preparation method of a suspension containing iron ions for inhibiting postharvest fruit putrefactive pathogenic bacteria, which comprises the following steps:

(1) activating a Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) strain on a plate culture medium, selecting a single colony, inoculating the single colony into a test tube containing an LB liquid culture medium, and culturing at 37 ℃, 180 and 220rpm for 12-16h to obtain a Bacillus amyloliquefaciens seed culture solution;

the Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13466 and the preservation date of 2016, 12 and 19 days, and is recorded in the Chinese patent application CN201710100745.9 which is earlier applied by the applicant;

(2) inoculating the seed culture solution into LB liquid culture medium according to the volume ratio of 1.0-2.0%, culturing at 37 deg.C for 4-6h, and centrifuging the fermentation liquid at 20-30 deg.C and 5000-10000g for 2-6min to obtain thallus.

(3) And (3) re-suspending the thallus obtained in the step (2) by using an LB liquid culture medium containing iron ions to obtain an iron ion-containing bacteria suspension.

Preferably, in the step (3), the LB liquid culture medium containing iron ions is used for resuspension until the concentration of the B4 bacterial suspension is 1X 10⁴-1×10⁷CFU/mL。

Preferably, the concentration of the iron ions in the bacterial suspension is 0.3-1.0 mM.

The invention provides the iron ion-containing bacterial suspension for inhibiting postharvest fruit putrefactive pathogenic bacteria, which is prepared by the preparation method.

The invention further provides application of the suspension containing the iron ions to inhibition of postharvest fruit putrefactive pathogenic bacteria, and the suspension is characterized in that the iron ions exist in the forms of ferric chloride, ferric sulfate and ferric nitrate.

Preferably, the fruit is pear, loquat, cherry tomato.

Preferably, the postharvest fruit spoilage pathogens include: penicillium expansum, Botrytis cinerea.

The invention is applied to the biological control of postharvest fruit putrefactive pathogenic bacteria in any one of the following modes:

mode A: the concentration is 1 x 10⁴-1×10⁷Spraying the CFU/mL suspension containing the iron ions on the surfaces of the picked fruits to wet the surfaces of the fruits; after treatment, standing and airing at room temperature and under the condition that the relative humidity is 40% -90%;

mode B: soaking picked fruit in 1 × 10⁴-1×10⁷Soaking in CFU/mL iron ion-containing bacteria suspension for 0.5-2.5 min; after treatment, standing and airing at room temperature and under the relative humidity of 40% -90%.

In addition, compared with common chemicals and plant extracts in the field of biocontrol, the microbial fermentation product has the advantages of simple and easily obtained active ingredients, low price and the like.

The exogenous iron ions applied by the invention effectively enhance the understanding of the formation of the bacillus amyloliquefaciens biofilm and improve the inhibition capability on postharvest diseases of different fruits (such as loquats, crystal pears and cherry tomatoes). The biological control agent has the characteristics of safety and high efficiency, is environment-friendly, has an obvious biological control effect, has the advantages of simplicity, easiness in obtaining, low price and the like compared with common exogenous addition substances, and is favorable for promoting the popularization and the use of the biological control agent.

Drawings

FIG. 1 plate culture behavior of the strain B4 of the present invention;

FIG. 2 the effect of different concentrations of ferric chloride on biofilm of strain B4: (A) biofilm formation process, (B) biofilm biomass.

Detailed Description

The following examples of the present invention are described in detail, but the following examples are not intended to limit the scope of the present invention.

Example 1: promoting effect of ferric chloride on biofilm formation by bacillus amyloliquefaciens B4:

activating a Bacillus amyloliquefaciens B4(Bacillus amyloliquefaciens B4) strain on a plate culture medium, selecting a single colony, inoculating the single colony into a test tube containing an LB liquid culture medium, carrying out shake culture for 12h in a shaking table at 37 ℃ and the rotating speed of 180rpm, transferring 1mL of bacterial liquid into 50mL of the LB liquid culture medium, and continuously culturing until OD is achieved₆₀₀Is 0.5. FIG. 1 shows the plate culture behavior of the strain B4 according to the present invention.

The plate medium of this example is LB solid medium, which consists of: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 20g/L agar, adjusting pH to 7.4 with 0.1mol/L NaOH, and sterilizing at 121 deg.C for 20 min.

The LB liquid medium in this example consisted of: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, adjusting pH to 7.4 with 0.1mol/L NaOH, and sterilizing at 121 deg.C for 20 min.

And (3) centrifuging the fermentation liquor at 6000g and 27 ℃ for 5min, adding an equal volume of sterile LB liquid culture medium into a centrifugal tube filled with thallus precipitates, uniformly oscillating, and centrifuging and eluting again to obtain the thallus.

Re-suspending the bacteria with sterile LB liquid culture medium until the concentration of the bacillus amyloliquefaciens B4 bacterial suspension is 1 × 10⁷CFU/mL, spare.

In a 96-well U-shaped microplate, 150. mu.L of LB liquid medium containing 0.2mM, 0.5mM, 1.0mM ferric trichloride was added per well, and the negative control group was an equal amount of sterile LB liquid medium. Inoculating the treated bacterial suspension into 96-well plate at a ratio of 1:15, i.e. adding 10 μ L B4 bacterial suspension per well, controlling suspensionThe liquid concentration is 1 × 10⁶CFU/mL, 3 replicates per treatment, plates were placed in a 37 ℃ incubator and biofilm phenotype was observed periodically and photographed.

Standing for 24h, sucking out the culture solution under the biological tunica, washing the microporous plate with 160 μ L distilled water for 2-3 times, adding 160 μ L0.1% (w/v) crystal violet solution for dyeing, and removing the crystal violet dyeing solution after 20 min. Washing the microporous plate with distilled water until the microporous plate is no longer purple in clear water, adding 160 muL of 30% acetic acid solution to completely dissolve the biofilm, diluting by 20 times, transferring to an enzyme label plate, measuring the light absorption value at 560nm, and reflecting the biofilm biomass by multiplying the light absorption value by the dilution times.

As shown in FIG. 2, the biofilm-forming ability of Bacillus amyloliquefaciens B4 was enhanced and the biofilm biomass increased with the increase of the concentration of the added ferric chloride.

Example 2: the ferric chloride improves the effect of the bacillus amyloliquefaciens B4 on inhibiting penicillium expansum in pear, cherry tomato and loquat fruits:

preparation of a suspension of spores of Penicillium expansum (Penicillium expansum): selecting a ring of pathogenic fungi with inoculating ring, inoculating into culture medium of PDA test tube slant, culturing at 25 deg.C for 7 days, scraping off part of spores, suspending in sterile normal saline, counting with blood count plate to determine spore concentration, and diluting with sterile normal saline to 1 × 10⁵conidia/mL for use.

Preparation of PDA culture medium: boiling 200g peeled potato in appropriate amount of distilled water for 20min, cooling at room temperature, filtering with 8 layers of gauze, adding 20g glucose and 1.5% (w/v) agar into the filtrate, diluting with distilled water to 1L, and sterilizing at 115 deg.C for 30 min.

PDA medium was sterilized, cooled to 50-60 deg.C, ferric chloride (final concentration 0, 0.2mM, 0.5mM, 1.0mM) was added at the corresponding concentration, 15mL of medium was poured into each plate, 5. mu.L of the above penicillium expansum spore suspension was added at the center of the plate after solidification, cultured in an incubator at 25 deg.C and the growth diameter was measured periodically, three replicates per treatment. As shown in Table 1, it can be seen from Table 1 that the growth diameter of Penicillium expansum is not so different when ferric chloride is added at a concentration of up to 1 mM. Analysis of variance showed some difference in the addition of 1mM ferric chloride from the control, but this was due to the fact that when the spore suspension was added centrally to the plate, the droplets showed a difference in growth diameter due to the smaller base diameter caused by surface tension created by the higher ferric chloride concentration. The growth rate of the penicillium expansum is basically consistent among different concentrations of ferric chloride, and the ferric chloride in the tested concentration does not inhibit the growth of the penicillium expansum.

The B4 bacterial suspension was prepared in the same manner as in example 1.

Preparation of Fe-B4 bacterial suspension: the B4 bacterial suspension was diluted 10-fold with LB liquid medium containing 0.5mM ferric chloride at a concentration of 1X 10⁶CFU/mL。

27 crystal pears, 171 cherry tomatoes and 81 loquats in the same batch, which are not mechanically damaged and infected and have basically consistent sizes and maturity, are selected. Soaking in 0.2% sodium hypochlorite for 2min for sterilization, and washing. And (3) punching holes on the same part of the surface of each pear fruit by using a sterilized puncher, wherein the hole diameter is 5mm, the depth is 3mm, and each pear fruit is punched with 3 holes. Punching the same part of the surface of each loquat or cherry tomato fruit by using a sterilized puncher, wherein the hole diameter is 3mm, the depth is 1.5mm, and each fruit is punched with 1 hole. The crystal pears are 9 in one group, and each group is processed in parallel. The loquat is 9 as one basket, the cherry tomato is 19 as one basket, one group processes three baskets, and each group processes three groups in parallel.

The first group of crystal pears is added with 20 mu L of LB liquid culture medium in the holes, the second group is added with 20 mu L B4 bacterial suspension in the holes, and the third group is added with 20 mu L of Fe-B4 bacterial suspension in the holes. 10 mu L of LB liquid culture medium is added into the first group of loquat and cherry tomato, 10 mu L B4 bacterial suspension is added into the second group of holes, and 10 mu L of Fe-B4 bacterial suspension is added into the third group of holes. Left at room temperature until no liquid was evident in the wells (about 2 h).

After air drying, inoculating 20 mu L of penicillium expansum spore suspension into the wound of each crystal pear, inoculating 10 mu L of penicillium expansum spore suspension into the wound of each loquat or cherry tomato, sealing by using a PE plastic preservative film after completely air drying, and storing the fruits at normal temperature (25 ℃). The fruit incidence was observed every day from day 3, and the fruit wound incidence was counted, and the results were expressed as the average incidence (%).

The results of the experiments are shown in tables 2, 3 and 4. As can be seen from tables 3, 3 and 4, the incidence of LB medium blank group without Bacillus amyloliquefaciens B4 bacterial liquid in crystal pear, loquat and cherry tomato after inoculation of Penicillium expansum stored for 3 days at 25 ℃ is as high as 96.30%, 55.56% and 88.23%, and all the diseases occur on day 4. In the control group added with the bacterial liquid B4 and the group added with ferric chloride with a certain concentration on the basis, the loquat and cherry tomato begin to attack on the 4 th day, and the pear fruit does not begin to attack until the 5 th day, which shows that the bacillus amyloliquefaciens B4 obviously inhibits the attack of the pear fruit penicillium expansum. Compared with the B4 group, the incidence rate of the penicillium expansum treated by adding 0.5mM of ferric chloride is reduced by 18.52% on the 5 th day, the incidence rate of the loquats is reduced by 33.34% on the 4 th day, and the incidence rate of the cherry tomatoes is reduced by 20.00% on the 5 th day, so that the ferric chloride can greatly improve the incidence inhibition effect of the B4 on the penicillium expansum.

TABLE 1 growth diameter of Penicillium expansum treated with ferric chloride at different concentrations

TABLE 2 Effect of different treatments on Penicillium expansum in Pear fruits

TABLE 3 Effect of different treatments on the onset of Penicillium expansum in loquat fruits

TABLE 4 Effect of different treatments on the pathogenesis of Penicillium expansum in cherry tomato fruits

Example 3: the effect of bacillus amyloliquefaciens B4 on inhibiting botrytis cinerea in cherry tomato and loquat fruits is improved by ferric chloride:

preparation of a suspension of Botrytis cinerea (Botrytis cinerea) spores: inoculating a loop of mycelia on a Botrytis plate, inoculating into culture medium on PDA test tube slant, culturing at 25 deg.C for 7 days, scraping off part of spores, suspending in sterile normal saline, counting with blood count plate to determine spore concentration, and diluting with sterile normal saline to 1 × 10⁵conidia/mL for use.

The suspension of Fe-B4 was prepared as in example 2.

The cleaning treatment of loquat and cherry tomatoes was the same as in example 2.

Exogenous disease of loquat, cherry tomato in example 3 a suspension of Botrytis cinerea spores was used.

As shown in tables 4 and 5, the incidence rate of LB medium blank group without adding Bacillus amyloliquefaciens B4 strain in loquat and cherry tomato is already as high as 88.89% and 87.50% when stored for 3 days at 25 ℃ after inoculation of Botrytis cinerea, and the disease is totally developed on the 4 th day. The cherry tomatoes added with the Fe-B4 bacterial liquid started to develop on day 5, and compared with the B4 treated group, the incidence of the botrytis cinerea treated with 0.5mM ferric chloride was reduced by 55.56%, 44.44% and 33.33% on days 3, 4 and 5, respectively. The incidence of the cherry tomato botrytis cinerea treated by adding 0.5mM of ferric chloride is reduced by 7.14%, 8.57% and 23.33% on days 3, 4 and 5 respectively. The results show that the ferric chloride can obviously improve the disease inhibiting effect of B4 on botrytis cinerea.

TABLE 4 Effect of different treatments on the onset of Botrytis cinerea in loquat fruits

TABLE 5 Effect of different treatments on the pathogenesis of Botrytis cinerea in cherry tomato fruits

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.