CN107267423B

CN107267423B - Bacillus amyloliquefaciens L-1 for antagonizing pear diseases and application thereof

Info

Publication number: CN107267423B
Application number: CN201710632072.1A
Authority: CN
Inventors: 孙平平; 崔建潮; 贾晓辉; 王文辉
Original assignee: Fruit Tree Institute of CAAS
Current assignee: Fruit Tree Institute of CAAS
Priority date: 2017-07-28
Filing date: 2017-07-28
Publication date: 2020-09-01
Anticipated expiration: 2037-07-28
Also published as: CN107267423A

Abstract

The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L-1 for antagonizing pear diseases, which belongs to the Bacillus subtilis with the preservation number as follows: CGMCC No. 14373. Research results show that the strain L-1 can effectively inhibit the expansion of the ring spot of pomes, cause hypha deformity of pathogenic bacteria, and has obvious antagonistic activity on the ring spot of pomes and common fungal diseases of apples and pomes. The strain L-1 can induce the activity expression of fruit resistance related enzymes (POD, CAT), reduce the accumulation of malondialdehyde in fruits and delay the fruit aging; l-1 has no damage to fruit quality and can increase VC content in fruits; l-1 can be successfully and rapidly propagated in fruit wounds; the strain has the advantages of salt tolerance, stable activity of active secondary metabolites, ultraviolet irradiation resistance, acid and alkali resistance, high temperature resistance, metal ions and the like, and has great application potential.

Description

Bacillus amyloliquefaciens L-1 for antagonizing pear diseases and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to bacillus amyloliquefaciens and application thereof.

Background

China is the biggest pear production and export country in the world, in 2016, the total output of pears in China reaches 1930 ten thousand tons and accounts for 75% of the total output in the world, and the export quantity gradually surpasses European Union and Argentina and becomes the biggest export country of pears in the world (Sun Ping and Wang Wenhui, 2016); however, the production management level of the pear orchard in China is backward (Zhang Shao Ling, 2013), diseases are serious, pesticide residues exceed the standard (Wang Tianli, 2015), and the average yield, quality and price of fruits are obviously lower than those of developed countries (Wang Wen Hui et al, 2014). Along with the increase of the output of Chinese pears, the improvement of the fruit quality, the enhancement of the disease resistance of trees, the reduction of the occurrence of diseases and the application of chemical pesticides are main measures for realizing the loss reduction and income increase of the pear industry and the enhancement of the international competitiveness.

Pear is infected by a plurality of diseases such as pear ring rot, gray mold, penicilliosis and the like in the production and storage processes of pears, wherein the pear ring rot is caused by grape-seat fungus (Botryosphaeria berengiana), which damages fruits, branches and leaves, pathogenic bacteria B.berengiana invade from skin holes or wounds of branches or fruits in the young fruit period, the disease is intensively developed 40-60 days after picking (field road, etc., 2013), the yield of the fruits is reduced by about 25% every year, and the rotten fruit rate is as high as more than 80% in severe cases (wang yan, etc., 2008), so that serious loss is caused.

At present, the prevention and treatment of the ring rot of the pome are mainly implemented in the young fruit period, the middle fruit growth period and before harvesting by using an organophosphorus bactericide such as carbendazim, fosetyl-aluminum, thiophanate-methyl and the like and Bordeaux mixture and the like (Maguangdian, 2007), but the application of chemical agents seriously harms human health, pollutes the environment, easily causes the drug resistance of pathogenic bacteria, and severely limits the safety and export of fruits due to the problems of pesticide residue and the like caused by large-scale application (Ragsdale & Sisler, 1994), so that an efficient and pollution-free prevention and treatment method is urgently needed to be found.

The substitution of effective microorganisms for chemical bactericides for preventing and treating postharvest diseases has shown a huge application prospect, and the biological prevention and treatment of plant diseases has the advantages of diverse action mechanisms, safety, environmental protection, persistent effect, resistance reduction and the like, and becomes a main method for replacing chemical prevention and treatment (Cook, 1993). Biocontrol bacteria can inhibit plant diseases by secreting resistant substances, by parasitic action, by spatial and nutritional competition, and by inducing plants to develop disease resistance (palaaniandi et al, 2013).

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to provide a microorganism which can effectively antagonize pear diseases so as to solve the problems of serious pear diseases, high residue caused by chemical agent control and the like.

The technical scheme of the invention is as follows: bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L-1 for antagonizing pear diseases is Bacillus subtilis, is preserved in China general microbiological culture Collection center in 2017 in 30.06 months, and has the preservation address as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and the preservation numbers are as follows: CGMCC No. 14373.

The application of the strain L-1 or the metabolite thereof in preventing and treating the ring rot pathogen.

The application of the strain L-1 or metabolite thereof in preventing and treating pear scab, botrytis cinerea, brown rot and penicillium.

The application of the strain L-1 or the metabolite thereof in preventing and treating anthracnose and moldy heart of apple.

The application of the strain L-1 or the metabolite thereof in enhancing the peroxidase and catalase activity of pear.

The application of the strain L-1 or the metabolite thereof in reducing accumulation of malondialdehyde in fruits.

The application of the strain L-1 or the metabolite thereof in delaying fruit senescence in the pear preservation process.

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

1. according to the invention, a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L-1 is separated and identified, and research results show that the strain L-1 can effectively inhibit the expansion of pear ring spot diseases, so that pathogenic bacteria hypha malformation is caused, and the strain has obvious antagonistic activity on the pear ring spot diseases and common fungus diseases of apples and pears.

2. The strain L-1 can induce the activity expression of fruit resistance related enzymes (POD, CAT), reduce the accumulation of malondialdehyde in fruits and delay the fruit aging; l-1 has no damage to fruit quality and can increase VC content in fruits; l-1 can be successfully and rapidly propagated in fruit wounds; the strain has the advantages of salt tolerance, stable activity of active secondary metabolites, ultraviolet irradiation resistance, acid and alkali resistance, high temperature resistance, metal ions and the like, and has great application potential.

3. The popularization and application of L-1 provide more foundations for reducing the chemical agent residue of pomes and realizing green and safe fruit production. The biocontrol agent has the characteristics of persistence, stability, no pollution, safety to people and livestock, no residue, strong specificity and the like in the biological control of fruit diseases, and has the functions of protecting the environment and maintaining ecological balance.

Drawings

FIG. 1 is a test of the in-dish antagonistic activity of strain L-1 against Phyllostachys verticillata, wherein A is the in-dish antagonistic activity of strain L-1 against Phyllostachys verticillata and the effect on the growth of hyphae thereof, B is the normal hyphae of Phyllostachys verticillata, and C and D are hyphae malformation of Phyllostachys verticillata caused by strain L-1;

FIG. 2 shows the antagonistic activity of the strain L-1 against Ribes burejense, Penicillium and Botrytis cinerea;

in FIG. 3, A is 16S rDNA of strain L-1, B is gyrA sequence to establish phylogenetic tree, wherein the black dots represent the classification status of strain L-1;

FIG. 4 shows that the bacterial strain L-1 induces the enzymatic activities of POD and CAT of pome to slow down the accumulation of MDA;

FIG. 5 shows the permanent planting of Bacillus amyloliquefaciens L-1 in the wound of pear fruit during the storage at room temperature;

FIG. 6 shows the effect of strain L-1 on pear hardness, soluble solids, VC and titratable acid content;

FIG. 7 shows the activity of the secondary metabolites of strain L-1 in different acid-base, UV, high temperature, metal ions and protease-degrading enzymes.

Detailed Description

EXAMPLE 1 isolation of the strains

Rhizosphere soil of pear trees is collected in Xingsheng city of Liaoning province in 2015, 9 months, and bacteria in the soil are separated by using a dilution plate method (Herr, 1959). Weighing 10g of the soil sample subjected to drying treatment, fully grinding, transferring the ground soil sample into a triangular flask containing 90mL of sterile water, and fully oscillating the soil sample at the temperature of 4 ℃ and the speed of 120r/min for 30 min. Diluting the soil sample suspension step by step in a clean bench, sucking 100uL of the diluted solution, uniformly coating the diluted solution on an NBA (nutrient Broth agar) (3g of beef extract, 5g of peptone, 2.5g of glucose, 20g of agar powder and 1L of water) culture medium, culturing for 1d at 32 ℃, and picking single colonies to purify on the NBA culture medium. 90 single colonies after purification were transferred to new NBA medium and stored for future use.

Example 2 active Strain screening

The inhibitory activity of the 90 isolated strains of bacteria on pyricularia piricola was determined by the plate confrontation method (Khamna et al, 2009) as follows: respectively picking and culturing on PDA culture medium (potato extract powder 6g, glucose 20g, Agar powder 20g and distilled water 1L) for 5d, placing ring spot germ cake with diameter 6mm in the center of PDA flat plate, perforating at both sides 3cm away from the germ cake, removing culture medium, inoculating to NBA culture medium to grow bacteria cake to be screened for 2d, and placing at 25 deg.C for constant temperature culture. Each treatment was repeated 3 times, using plates without actinomycetes as controls, and observing and recording the diameter of the zone of inhibition when the plates were full of control colonies. The results are shown in FIG. 1: the strain L-1 has obvious antagonistic activity (A in figure 1) on Physalospora piricola, and the bacteriostatic circle preparation reaches more than 20 mm. Microscopic observation of the control hyphae of ring spot and the hyphae of ring spot around the antagonistic circle revealed that the hyphae treated with the strain L-1 exhibited swelling, deformity, and the like (C, D in FIG. 1) relative to the control hyphae (B in FIG. 1).

Example 3 controlling Effect of active Strain L-1 on apple and Pear common fruit postharvest diseases

The antagonism effect of L-1 on common pear fruit diseases such as pear scab, gray mold, brown rot, penicillium, apple anthracnose, mildew, and the like is determined by an Oxford cup method. The L-1 strain is transferred into 100ml NB culture solution and cultured for 2 days at 28 ℃ and 180 r/min. Inoculating pathogenic bacteria cake to be tested in the center of PDA plate, placing sterilized Oxford cups on both sides, adding 200ul L-1 fermentation liquid into the Oxford cup, adding NB culture solution without L-1 spore as control, culturing at constant temperature of 25 deg.C, and repeating for 3 times each treatment. The control growth dish was used to measure the growth diameter of the pathogenic bacteria, and the control effect was calculated using (control effect (%) ═ (1-treatment lesion diameter/control lesion diameter) × 100). The results are shown in Table 1, and the strain L-1 has significant antagonistic activity on Pyricularia nigra, Botrytis cinerea, Monilinia fructicola, Penicillium, colletotrichum malorum and Cordyceps myceliophthora, and has a wide antibacterial spectrum.

TABLE 1 in vitro control effect of the strain L-1 on common 6 fruit diseases of pear and apple

EXAMPLE 4 in vivo control of Pear disease by Strain L-1

The in vivo inhibitory activity of strain L-1 against Pear ring rot, Penicillium and Botrytis cinerea was determined by reference to the method of Sadeghian et al (2016), 3 times for each treatment, and 6 fruits were repeated.first, fruits were surface-sterilized by reference to the method of Zhang et al (2005). healthy fresh Huangguan pears were immersed in 2% sodium hypochlorite for 3min, rinsed with tap water and then air-dried, 5mm (diameter) × 3mm (depth) wounds were made on both sides of the equator of the pears with sterile punches, then 2d L-1 spore suspension was cultured in NB medium at 150rpm and 32 ℃ and diluted with sterile water to 1 × 10 d⁸CFU/ml spore suspension, 30. mu.l spore suspension was added to each wound, so that the same volume of NB medium was added as a control after the wound. The treated fruits are placed in a plastic box paved with gauze, sealed by a preservative film and subjected to moisturizing culture at 20 ℃. Inoculating the ring veins, gray mold and penicillium bacteria cake on the wound respectively, continuously culturing for 1d, removing the bacteria cake, continuously culturing in a moisture-preserving manner, and measuring the diameter of the lesion. The results are shown in figure 2, the inoculation of the L-1 strain can obviously inhibit the lesion extension of the pear ring rot, the penicillium and the gray mold, the prevention and treatment effect on the pear ring rot in 11 days after inoculation reaches 76.55%, the prevention and treatment effect on penicillium and gray mold in 25 days after inoculation reaches 77.47%, and the prevention and treatment effect on gray mold in 9 days after inoculation reaches 92.88%.

EXAMPLE 5 identification of Strain L-1

The observation and evaluation of the colony culture characteristics, morphological characteristics and utilization characteristics of lactose, galactose, xylose, maltose, starch, glucose and fructose of L-1 revealed that the strain L-1 may belong to bacteria and can utilize various carbon sources.

Extracting L-1 total DNA according to the method of an Ezup column type bacterial genome DNA extraction kit (SK8255), adopting universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3')/1492R (5'-GGTTACCTTGTTACGACTT-3') and Gyra-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3')/Gyra-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3'), respectively amplifying 16S rDNA and Gyra gene of the strain (Daibobo et al, 2016), 25 mul PCR reaction system comprises 0.2 mul of Premix Taq, 2.5 mul of 10 × buffer, 0.5 mul of each primer and 0.5 mul of Template DNA, filling sterile water to 25 mul, and carrying out PCR reaction under the conditions of 94 ℃ for 4min and 4min35 amplification cycles (94 ℃ 30S, 54 ℃ 45S, 72 ℃ 90S), 72 ℃ 10 min. After the reaction, 5L of the reaction solution was subjected to electrophoresis detection using 1% agarose gel, and the target band was recovered using a Sanprep column type DNAJ gel recovery kit (SK8131) and ligated to p^topoThe sequence was aligned using the MEGA5.0 software, the sequence was aligned using the Clustal W algorithm, phylogenetic trees were constructed using the neighborhood method (NJ), the alignment value was set to 1000, and the results of the evolution were 50% to show that the strain L-1 and Bacillus amyloliquefaciens CAB 946(HE617159) and FZB42(NR _075005) were clustered into one, and the homology was 99.1669% to 99.9% to construct Bacillus amyloliquefaciens CAYR-KC 946(HE617159) and FZB 16632 (NR _075005), respectively, based on the 16S rDNA evolutionary trees^T(AF272015) was clustered to one branch, and the homology reached 99.8% (B in FIG. 3), so that strain L-1 was identified as Bacillus amyloliquefaciens.

Example 6 Effect of Strain L-1 on fruit POD, CAT Activity and malondialdehyde content

Measurement of the Peroxidase (POD) and Catalase (CAT) enzymatic activities of fruits and the Malondialdehyde (MDA) with reference to Zhang et al (2016), yellow-crowned pears were disinfected and wounded, and inoculated with 50. mu.l of 1 × 10⁸CFU ml^-1The suspension of the L-1 spore or sterile water is subjected to moist culture at 20 ℃. Taking pear pulp 1cm around the wound at 0(2h), 1, 2, 3, 4 and 5d, freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C. The fresh sample amount of POD and CAT with a 1S-induced OD value change of 0.01 was defined as 1 enzyme activity unit (U) of POD and CAT, and U.kg was used as a result^-1S^-1Expressed as μmol per kg fresh sample (μmol. kg)^-1) Representing the malondialdehyde content, each assay was repeated 3 times. The results are shown in FIG. 4: the L-1 treatment significantly increased the crown pears relative to the controlThe POD and CAT enzyme activities of the fruits reach 25.56 times and 3 times of those of the control respectively 3 days after inoculation, and the activities are still higher than those of the control 5 days after inoculation. The level of malondialdehyde indicates the degree of senescence of the fruit, and the MDA content of the fruit after L-1 treatment was significantly lower in this study than the control treatment (C in FIG. 4). The results show that the strain L-1 can obviously induce the enzymatic activities of POD, CAT and the like related to fruit resistance, reduce the accumulation of MDA in fruits and delay the fruit senescence.

Example 7 colonization of Strain L-1 in fruit wounds

The method of example 3 is to sterilize and perforate the Huangguan pear fruit, add 30 mul L-1 bacterial strain spore suspension, culture at 20 ℃ with moisture retention, take 0d (2h), 1d, 2d, 3d, 4d, 5d, 6d of the inoculated Huangguan pear and 0.5g of pulp around the wound by 7mm perforator, grind and dilute the pulp to a certain concentration with sterile water, evenly coat the pulp on NBA culture medium, culture at 32 ℃ for 2d, calculate the clone number, show that the bacterial strain L-1 can rapidly propagate in the fruit wound, inoculate 2h with 5.075 × 10⁶CFU, increased to 6.886 × 10 at 3d culture⁸And the number of hours reaches 7.275 × 10 in 4d⁸CFU, 101.8 times higher than at inoculation and always kept high in storage, kept the number of clones at 6.933 × 10 for 6d after inoculation⁸And (4) CFU. As shown in fig. 5.

Example 8 Effect of Strain L-1 on fruit quality

The influence of L-1 on the fruit quality index was determined by reference to the method of Zhang et al (2008), and 15 fruits were selected for each determination. Soaking the 'Huangguan pear' fruit with the surface disinfected in a culture medium containing L-1 spore suspension for 30S, storing at 20 ℃, and periodically measuring the hardness, soluble solid matters, titratable acid and Vc content of the fruit. The pulp hardness was measured using a GS-15 fruit texture analyzer, and as a result, it was measured in kg. cm^-2The content of soluble solids in the fruit was measured by PR-101 α sugar breaking instrument, and the results were expressed as% the contents of titratable acid and Vc in the fruit were measured by Metrohm 808Titrando potentiometric titrator, the titratable acid was calibrated by NaOH, the Vc content in the fruit was calibrated by 2, 6-dichlorophenol indophenol, and the results were expressed as% in mg/kg^-1Fresh weight is expressed. The results show that the strain L-1 is applied to fruitThe quality is not damaged: the hardness of the fruit is maintained at 4.90 + -0.42 to 5.49 + -0.48 kg-cm^-2The content of soluble solid is between 9.85 plus or minus 0.70% and 11.01 plus or minus 0.82%, the Vc content of the crown pear fruit treated by the L-1 is higher than that of the control treatment, and the TA content is between 0.075 plus or minus 0.001% and 0.093 plus or minus 0.002% (figure 6).

EXAMPLE 9 evaluation of the stability of Strain L-1

The stability of the strain is the basic premise of ensuring the control effect, and the stability of the active biocontrol bacterium L-1 fermentation liquor is evaluated. The L-1 is placed in NB medium and cultured for 48h at the temperature of 28 ℃ at 180 r/min. The L-1 strain fermentation liquor is centrifuged for 10min at 8000rpm, and the supernatant is taken and filtered by a 0.22um filter membrane to obtain the fermentation liquor without spores. The pH stability, UV resistance, heat treatment, metal ion and protease degradation properties of the L-1 fermentation broth were determined by Oxford cup method, and the results are shown in FIG. 7.

pH stability: adjusting the pH value of the L-1 fermentation broth to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 with NaOH and HCl respectively, and measuring the activity change by an Oxford cup method; the result shows that the optimum pH of the strain L-1 fermentation filtrate is 6-7, the fermentation liquor is acid and alkali resistant, and the antagonistic activity of the strain L-1 on pathogenic bacteria still reaches more than 40% when the pH is 12.

Ultraviolet stability: placing L-1 strain fermentation liquid at 40cm under 20W ultraviolet lamp, irradiating for 10min, 20min, 30min, 40 min, 50 min, 60 min, 90 min, and measuring activity change with Oxford cup method with active crude extract as control; the result shows that the antagonistic activity of the L-1 fermentation liquor is obviously influenced within 2 hours of ultraviolet irradiation.

Thermal stability: treating the fermentation broth at 40, 50, 60, 70, 80, 90, 100 deg.C for 20min, cooling to room temperature, and determining activity change by Oxford cup method with room temperature treatment as control; the results show that the high temperature treatment has a significant effect on the activity of the L-1 fermentation broth.

Effect of different metal ions on compound activity: adding CaCl₂、ZnSO₄·7H₂O,CuSO₄·5H₂O、NaCl、MgSO₄·7H₂O、MnCl₂·4H₂O、FeSO₄·7H₂O, KCl to 1mAdding 100 mul of metal ion solution into 1000 mul of fermentation liquor to prepare 0.1mol/l of solution, shaking and uniformly mixing, and treating in water bath at 37 ℃ for 1 h. The effect of different metal ions on the activity of the fermentation liquor is determined by an Oxford cup method. The result shows that the strain L-1 fermentation liquor is resistant to heavy metals, and the antagonistic activity of the strain L-1 fermentation liquor on pathogenic bacteria in 0.1mol/L metal ions still reaches more than 50%.

Protease degradation stability: trypsin, pepsin and proteinase K are prepared into a 1mg/ml solution, the solution reacts for 1h at 37 ℃, then the solution is treated at 80 ℃ for 30min and immediately cooled at 4 ℃, and the antagonistic activity of the solution is measured, so that the result shows that the antagonistic activity of the L-1 fermentation broth product is not influenced by the treatment of the trypsin, the pepsin and the proteinase K, and active compounds are presumed not to belong to proteins.

Strain L-1 salt tolerance: respectively adding 0.1, 0.5, 1, 2, 5 and 10% NaCl into NB culture solution, transferring L-1 into salt-containing culture solution with different concentrations, culturing at 28 deg.C at 180r/min, and measuring OD value at 600 nm. The results show that: the strain L-1 can still grow in 10% NaCl, and the strain is salt-tolerant.

The results show that the strain L-1 has good environmental adaptability and stress resistance, and lays a good foundation for commercial production of the strain L-1.

EXAMPLE 10 preparation of lyophilized powder of Strain L-1

Transferring 100 μ L of L-1 stored at-80 deg.C in 20% glycerol into 100ml NB culture medium, culturing at 28 deg.C for 48 hr at 180r/min to obtain L-1 mother solution. Transferring 1mL of mother liquor into a new 100mL of NB culture solution, continuously culturing at 180r/min and 28 ℃ for 2d, centrifuging the cultured L-1 strain at 4 ℃ and 6000rpm for 10min, collecting precipitate, using 10% skimmed milk powder as a protective agent, and freeze-drying in vacuum for 20 h. The freeze-dried powder is stored at 4 ℃. The survival rate of the spores is determined by a dilution method after the freeze-dried powder is stored for 6 months, and the result shows that the survival rate of the strain L-1 still reaches 91.25 percent.

And (4) conclusion:

according to the invention, a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L-1 is separated and identified, and research results show that the strain L-1 can effectively inhibit the expansion of ring rot of pome fruits, cause hypha deformity of pathogenic bacteria, has obvious antagonistic activity on the ring rot of the pome fruits and common fungal diseases of apples and pomes, can induce the activity expression of related enzymes (POD, CAT) for resistance of the fruits, reduce the accumulation of malonaldehyde in the fruits and delay the senescence of the fruits; l-1 has no damage to fruit quality and can increase VC content in fruits; l-1 can be successfully and rapidly propagated in fruit wounds; the strain has the advantages of salt tolerance, stable activity of active secondary metabolites, ultraviolet irradiation resistance, acid and alkali resistance, high temperature resistance, metal ions and the like, and has great application potential. The popularization and application of the L-1 provide more foundations for reducing the chemical agent residue of the pome and realizing green and safe fruit production. The biocontrol agent has the characteristics of persistence, stability, no pollution, safety to people and livestock, no residue, strong specificity and the like in the biological control of fruit diseases, and has the functions of protecting the environment and maintaining ecological balance.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

Claims

1. The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) L-1 for antagonizing the pear diseases is a Bacillus, and the preservation number is as follows: CGMCC number 14373.