CN110724643A - Penicillium citrinum with disease prevention and growth promotion functions, microbial inoculum and application thereof - Google Patents
Penicillium citrinum with disease prevention and growth promotion functions, microbial inoculum and application thereof Download PDFInfo
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- CN110724643A CN110724643A CN201911129054.7A CN201911129054A CN110724643A CN 110724643 A CN110724643 A CN 110724643A CN 201911129054 A CN201911129054 A CN 201911129054A CN 110724643 A CN110724643 A CN 110724643A
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- penicillium citrinum
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- penicillium
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- microbial inoculum
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Abstract
The invention relates to a Penicillium citrinum strain with growth promoting effect and broad-spectrum disease preventing effect and a microbial inoculum thereof, wherein the Penicillium citrinum is Penicillium citrinum (Penicillium aurantiogenum) 44M-3 which is preserved in China center for type culture collection with the preservation number: CCTCC NO: m2019768. The blue mold strain of orange gray is used to prepare the high spore powder microbial inoculum, when in use, the high spore powder is resuspended in sterile water of 0.05% (v/v) Tween 80, and spore suspension liquid with different concentrations is dipped in roots or irrigated to treat the roots of the plants. The penicillium citrinum has good inhibition effect on pathogenic fungi on various crops; the penicillium citrinum high-spore powder suspension has the effect of promoting the growth of roots and plants of corn, wheat, cotton, sesame, cucumber and tobacco; meanwhile, the microbial inoculum has good biological safety, and biological preparations do not generate chemical and agricultural chemicals common problems such as drug resistance, pesticide residue, environmental pollution and the like, are healthy to people and livestock and environment-friendly, and meet the requirements of ecological agriculture and environmental protection.
Description
Technical Field
The invention relates to penicillium citrinum capable of preventing and treating various diseases and having a growth promoting effect and application thereof, and belongs to the technical field of microorganisms.
Background
The disease control of crops in China depends on chemical pesticides for a long time, so that the pesticide is used in excess, environmental pollution and food pesticide residue are possibly caused, and potential harm is brought to human health. The use of microbial agents and their metabolites, which are harmless to the human body and the ecological environment, can reduce the application of chemical pesticides, and has become a development direction worldwide. The biopesticide has the advantages of environmental friendliness, safety to people and livestock, long acting, no residue and the like, and is favored in controlling the occurrence and the spread of diseases.
The biological control microorganisms which are researched more at present are bacillus, pseudomonas, saccharomycetes, streptomyces, trichoderma and the like. The reports of the penicillium as the biocontrol bacterium for preventing and treating plant diseases are less, and researches show that the paecilomyces lilacinus strain has a certain control effect on the occurrence and spread of cotton wilt; the intracellular and exopolysaccharides of the paecilomyces lilacinus strain NH-PL-03 have obvious inhibition effects on the hypha growth and spore germination of fusarium oxysporum.
Regarding the report of penicillium citrinum, mainly the penicillium citrinum is taken as a fermentation strain, researches show that penicillium citrinum is a dominant strain for producing lipase and a dominant population for producing alpha-amylase in the fermentation process of soybean paste, and no report of systematic research on growth promotion and disease prevention effects and application of penicillium citrinum is reported.
Disclosure of Invention
The invention provides a penicillium citrinum strain with growth promoting effect and broad-spectrum disease preventing effect, and a microbial inoculum and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
the penicillium citrinum is penicillium citrinum (penicillium aurantium) 44M-3 which has been preserved in China center for type culture collection and has the preservation address: wuhan university collection center, accession number: CCTCC NO: m2019768, date of deposit: 09 and 30 days 2019.
A method for preparing a high sporopollen microbial inoculum by using penicillium citrinum strains comprises the following steps:
inoculating penicillium citrinum to a PDA culture medium, alternately culturing in a constant-temperature incubator with 25 ℃ and 8h of illumination/16 h of darkness for 5-7d in a light-dark manner, eluting spores by using 0.05% (v/v) Tween 80 sterile water to form a spore suspension, subpackaging PDB culture solution into 300mL triangular bottles, subpackaging 200mL each bottle, inoculating 5mL of the spore suspension to each bottle, culturing at 180r/min at 28 ℃ for 4d in a shaking way, and taking the spore suspension as a seed solution for solid fermentation culture for later use;
weighing 1500g of wheat, soaking in distilled water for 8h, taking out, draining, and packaging into fungus culture bags at 121 deg.C for 30min, each bag containing 100g of wheat; inoculating 10mL of seed solution into each bag, spreading to make the wheat uniformly infiltrate the seed solution, and culturing in a dark constant-temperature incubator at 28 ℃ for 7-10 days;
spreading the solid fermentation product in an enamel tray paved with a layer of sterile paper when the solid fermentation culture medium produces spores sufficiently, covering a layer of sterile paper, placing the enamel tray in a glass window capable of being irradiated by sunlight for airing for 5-7 days, collecting spore powder by using a BFQ100 fungal spore separator after airing, and forming a high-spore-powder microbial inoculum, wherein the spore content of penicillium citrinum in the high-spore-powder microbial inoculum is 1.96 multiplied by 109CFU/g。
The PDA culture medium: peeling 200g of potatoes, cutting into blocks, putting the cut potatoes into distilled water, boiling for 10min, filtering through 6 layers of gauze, adding 20g of glucose and 15-20 g of agar into filtrate, adding distilled water to a constant volume of 1000mL, and sterilizing for 30min at 121 ℃;
the PDB culture solution is as follows: peeling potato 200g, cutting into pieces, putting into distilled water, boiling for 10min, filtering with 6 layers of gauze, adding glucose 20g into the filtrate, adding distilled water to reach a constant volume of 1000mL, and sterilizing at 121 ℃ for 30 min.
The application method of the high spore powder microbial inoculum comprises the steps of resuspending high spore powder with 0.05% (v/v) Tween 80 sterile water, counting by using a blood counting chamber to adjust the concentration of spores, and dipping roots or irrigating roots with spore suspension liquid with different concentrations to treat the roots of plants.
A culture solution for producing bacteriostatic active substances by penicillium citrinum strains is prepared from the following components in percentage by weight:
30g of sucrose, 5g of peptone, 0.5g of KCl and MgSO4·7H2O 0.5g，FeSO4·7H2O0.01 g and distilled water 1000 mL.
The application of the penicillium citrinum in inhibiting the growth of pathogenic bacteria of wheat, tobacco, sesame, rehmannia and sweet osmanthus diseases is disclosed.
The application of the penicillium citrinum in promoting the growth of corns, wheat, cotton, sesame, cucumbers and tobaccos.
Application of the penicillium citrinum in preventing and treating sesame wilt is provided.
The method for preventing and treating sesame wilt by using penicillium citrinum comprises preparing spore suspension of penicillium citrinum strain with 0.05% (v/v) Tween 80, wherein the spore concentration is 1 × 107CFU/mL; and (3) irrigating roots of the sesames by using the spore suspension at the seedling stage of the sesames.
The invention has the beneficial effects that:
1. the invention provides a Penicillium biological agent with strong growth promoting capability and wide disease prevention spectrum, and the Penicillium aurantiogenum is identified as Penicillium citrinum through morphological identification and sequence evolution analysis.
2. The penicillium citrinum has good inhibition effect on pathogenic fungi on various crops. The test shows that: the bacteriostatic agent has good bacteriostatic action on pathogenic fungi on various crops of sesame, tobacco, wheat, sweet osmanthus and rehmannia, and the average bacteriostatic rate reaches 59%. The moniliforme has the highest bacteriostasis rate (75.00%), and then the rhizoctonia cerealis (69.34%), fusarium oxysporum (from sesame, 63.67%) and nigrospora sphaerica (63.64%) are sequentially arranged, so that the penicillium citrinum isolate has broad-spectrum bacteriostasis activity.
3. The high-spore powder microbial inoculum of penicillium citrinum prepared by the invention has the spore content of 109CFU/g; the high spore powder suspension liquid has promoting effect on the growth of corn, wheat, cotton, sesame, cucumber and tobacco root and plant.
4. The spore suspension obtained by the penicillium citrinum strain can effectively reduce the morbidity of the wilt, has the prevention effect on the wilt of sesame up to 85.25 percent, and has the prevention effect equivalent to that of a commonly used chemical pesticide carbendazim 1000X diluent for preventing and treating the wilt.
5. The penicillium citrinum strain can dissolve various insoluble inorganic phosphorus, secretes siderophin and IAA, has the IAA yield of 29.3 mu g/mL, and can secrete protease, cellulase and beta-1, 3-glucanase.
6. After the screened penicillium citrinum is challenged to inoculate corn, wheat, cotton, sesame, cucumber and tobacco, 6 crops can normally grow, and no disease symptoms occur on roots and plants, so that the penicillium citrinum has no pathogenicity on the crops, has good biological safety, does not generate common chemical and pesticide problems such as drug resistance, pesticide residue, environmental pollution and the like in a biological preparation, is healthy to human and livestock, is environment-friendly, and meets the requirements of ecological agriculture and environmental protection.
Drawings
FIG. 1 shows the results of the co-culture of strain 44M-3 with a plate of 14 pathogenic fungi, where CK represents the control.
FIG. 2 is a diagram showing the control effect of the strain 44M-3 on sesame wilt.
FIG. 3 is a graph showing the results of the measurement of the yield of strain 44M-3 IAA.
FIG. 4 is a carbon nitrogen source optimization diagram of a fermentation culture solution of the strain 44M-3.
FIG. 5 is a graph of morphological characteristics and microscopic characteristics of strain 44M-3, in which "+" indicates the front of the plate, "-" indicates the back of the plate, A and B are conidiophores, and C is a conidiophore.
FIG. 6 is a phylogenetic dendrogram of the β -tubulin and Calmodulin sequences of strain 44M-3, wherein Panel A is the dendrogram of the β -tubulin sequences and Panel B is the dendrogram of the Calmodulin sequences; eupenicillium osmophilum in A is an ectogroup; in the B, Eupenicillium sinapicum is an outer group; the numbers in parentheses represent the accession numbers of the strains at NCBI.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Experimental materials:
the strain source is as follows: penicillium citrinum 44M-3, isolated from a soil sample from the Bianchi valley of Mianchi city, Sanmenxia, Henan province.
The Penicillium citrinum is Penicillium citrinum (Penicillium aurantigresum) 44M-3, which is preserved in China center for type culture Collection with the preservation address: wuhan university collection center, accession number: CCTCC NO: m2019768, date of deposit: 09 and 30 days 2019.
Bengal red medium: peptone 5g, glucose 10g, KH2PO41g，MgSO4·7H20.5g of O, 100mL of 1/3000(w/v) Bengal solution, 0.1g of chloramphenicol, 15-20 g of agar, 1000mL of distilled water and pH 7.2 +/-0.2.
Charles Agar (CA): chashi concentrate 10mL, K2HPO41g, 30g of cane sugar, 17.5g of agar and 1000mL of distilled water.
Charles yeast extract agar (CYA): k2HPO41g, 10mL of Chashi concentrated solution, 5g of yeast extract, 30g of sucrose, 15g of agar and 1000mL of distilled water.
Wort agar (MEA): malt Extract (Malt Extract)20g, peptone 1g, glucose 20g, agar 15g, distilled water 1000 mL.
25% glycerol nitrate agar (G25N): k2HPO40.75g, 7.5mL of Chashi concentrated solution, 3.7g of yeast extract, 250g of glycerol (analytically pure), 12g of agar and 750mL of distilled water.
Chashi concentrated solution: NaNO330g，KCl 5g，MgSO4·7H2O 5g，FeSO4·7H2O 0.1g，ZnSO4·7H2O0.1g，CuSO4·5H2O0.05 g and distilled water 100 mL.
PDA culture medium: peeling 200g of potatoes, cutting into blocks, putting the cut potatoes into distilled water, boiling for 10min, filtering through 6 layers of gauze, adding 20g of glucose and 15-20 g of agar into filtrate, adding distilled water to a constant volume of 1000mL, and sterilizing for 30min at 121 ℃. The PDB culture solution is PDA without agar.
Phosphorus dissolving capacity detection culture medium: glucose 10g, MgSO4·7H2O 0.3g，(NH4)2SO40.5g，NaCl 0.2g，KCl 0.2g，MnSO40.03g，FeSO4·7H200.01 g, yeast extract 0.5g, phosphorus source 5g, and distilled water 1000 mL. Respectively with Ca3(PO4)2、Zn3(PO4)2And hydroxyapatite as a phosphorus source, preparing 3 kinds of phosphorus dissolving capacity detection culture media, wherein the pH value of the 3 kinds of phosphorus dissolving capacity detection culture media is 7.0 +/-0.2, sterilizing at 121 ℃ for 30 min. Inoculating the strain 44M-3 toAnd (3) observing whether a transparent ring is generated on the phosphorus dissolving capacity detection culture medium, wherein the generated transparent ring is soluble.
Chitinase assay medium: colloidal chitin 5.0g, MgSO4·7H2O 0.5g，FeSO4·7H2O 0.01g，KH2PO40.3g，K2HPO40.7g，ZnSO4·7H20.001g of O, 20g of agar and 1000mL of distilled water, and the pH value is 7.2-7.4. And (3) detection: and (3) inoculating the strain 44M-3 on a chitinase detection culture medium, standing at 28 ℃ for 5 days, observing whether a transparent ring exists or not, and if the transparent ring is generated, generating chitinase.
Carboxymethyl cellulase detection medium: sodium carboxymethylcellulose 5.0g, MgSO4·7H2O 0.1g，(NH4)2SO40.5g，K2HPO40.25g, agar 20g, and distilled water 1000 mL. And (3) detection: the strain 44M-3 is spotted on a carboxymethyl cellulose culture medium, the culture is carried out for 5d at the temperature of 28 ℃, 1mg/mL Congo red is used for dyeing for 20min, 1mol/L NaCl is used for soaking for 15min, Congo red attached to the surface is washed by water, whether a transparent ring is generated around is observed, and if the transparent ring is generated, the cellulase is generated.
And (3) a casein enzyme detection culture medium: solution A: 5g of skimmed milk powder and 50mL of distilled water; and B, liquid B: agar 1.5g, distilled water 50 mL. And (3) separately sterilizing the solution A and the solution B, cooling to 45-50 ℃, mixing the two solutions, pouring the mixture into a flat plate to obtain a casein flat plate, and inverting the flat plate overnight to dry the surface water for later use. And (3) detection: the strain 44M-3 is spotted on a casein culture medium, and is cultured for 5d at 28 ℃, and whether a transparent ring is generated or not is observed, so that the transparent ring is generated, namely, protease is generated.
Beta-1, 3-glucanase assay medium: laminarin 0.005g, NaNO33.0g，KH2PO41.0g，KCl0.5g，MgSO40.5g，FeSO4·7H20.01g of O, 0.04g of Congo red, 20g of agar and 1000mL of distilled water, and the pH value is natural. And (3) detection: the strain 44M-3 is inoculated on a beta-1, 3-glucanase culture medium, the culture is carried out for 5 days at the temperature of 28 ℃, if laminarin is hydrolyzed, the red color disappears, and a colorless hydrolysis area appears on a flat plate.
And (3) detecting a siderophore culture medium:
preparing a detection solution (A): 60.5mg of Chromenium S was added to 50mL of deionized water, followed by 10mL of iron solution (1 mMFeCl)3·6H2O0.027 g, 10mM HCl 8.33 μ L) were mixed and the mixture was slowly added to the CTAB solution (72.9mg HDTMA added to 40mL water) with stirring; preparing a mixed solution (B): 750mL of water, 15g of agar, 30.24g of Pipes (1,4-Piperazinediethanesulfonic acid), 12g of 50% NaOH solution (w/w). Sterilizing at 121 deg.C for 15 min. And (3) slowly adding the A into the B along the inner wall of the glass while stirring, so as to avoid foaming.
Detecting siderophore: and (3) pouring 10mL of detection culture medium into a flat plate, cutting along a midline after the flat plate is solidified, removing half of the detection culture medium, adding 5mL of PDA culture medium, inoculating the strain 44M-3 on the PDA growth culture medium after solidification, approaching the midline position of the two culture media as far as possible, and observing whether the detection culture medium has color change.
Example 1 isolation of Strain 44M-3
The separation method comprises the following steps: drying the soil sample at room temperature, grinding and sieving, weighing 10g of ground and sieved soil sample, adding 90mL of sterile water, and fully oscillating to obtain a soil suspension, namely 10 of the soil sample-1Then shaking the soil suspension evenly and diluting the soil suspension to 10 degrees in a gradient way-2、10-3、10-4And 10-5And coating 100 mu L of diluent with different gradients on a Bengal red culture medium, carrying out inverted culture in a constant temperature incubator at 28 ℃ for 48h until bacterial colonies grow out, selecting a single bacterial colony to culture on a PDA culture medium for 5d, selecting a hypha block at the edge of the bacterial colony, transferring the hypha block to a new PDA plate, and repeating the transfer for 3 times to obtain a pure culture of the isolate.
Example 2 inhibitory Effect of the Strain 44M-3 on 14 pathogenic fungi on 5 plants
Plate co-culture method: inoculating strain 44M-3 to PDA culture medium plate, culturing for 8-10d, eluting spore with 0.05% (v/v) Tween 80 solution when 44M-3 produces large amount of conidia, and preparing spore suspension (2 × 10)7CFU/mL)；
Coating a PDA (personal digital Assistant) plate with 44M-3 spore suspension, culturing at 28 ℃ for 12h, beating a fungus cake with a 6-mm-diameter puncher, transferring the fungus cake to a symmetrical position which is 0.5mm away from the edge of a new PDA plate, inoculating pathogenic bacteria in the center of the plate, wherein the pathogenic bacteria are respectively 6 pathogenic bacteria on sesame, fusarium oxysporum, fusarium solani, helminthosporium sojae, ascochyta phaseoloides, rhizoctonia solani, polyspora polystictus and nigrospora sphaerica, 4 pathogenic bacteria on tobacco, moniliforme, phytophthora capsicum, fusarium oxysporum, rhizoctonia cerealis, plasmodiophora cinerea and rhizoctonia digitalis. Inoculating 44M-3 and 14 kinds of pathogenic bacteria, placing at 28 deg.C, dark culturing, measuring the size of the inhibition zone and the colony diameter of the pathogenic bacteria when the control plate is full of bacteria, and calculating the inhibition rate.
Bacteriostatic ratio (%) (control colony diameter-treated colony diameter) × 100/(control colony diameter-cake diameter)
As can be seen from Table 1 and FIG. 1, in the plate co-culture test, the strain 44M-3 has good bacteriostatic activity on 14 pathogenic bacteria to be tested, the width of a bacteriostatic zone is more than 10mm, and the average bacteriostatic rate on the 14 pathogenic bacteria reaches 59%, wherein the bacteriostatic rate on moniliforme is the highest (75.00%), and then the bacterial strains are rhizoctonia cerealis (69.34%), fusarium oxysporum (sesame) (63.87%) and nigrospora sphaera (63.64%) in sequence. The isolate 44M-3 has broad-spectrum bacteriostatic activity.
TABLE 1 bacteriostatic Activity of the Co-culture of Strain 44M-3 plates
Example 3 preparation of Strain 44M-3 high spore powder
Inoculating Penicillium citrinum on a PDA culture medium, alternately culturing in a constant temperature incubator at 25 deg.C under 8h light/16 h dark for 5-7d, and eluting spores with 0.05% (v/v) Tween 80 sterile water to form spore suspension; and (3) subpackaging the PDB into 300mL triangular flasks, wherein 200mL of PDB is filled into each flask, 5mL of spore suspension is inoculated into each flask, and the mixture is subjected to shake culture at the temperature of 28 ℃ for 4d at 180r/min to serve as seed liquid for solid fermentation culture.
Weighing 1500g of wheat, soaking in distilled water for 8h, taking out, draining, and packaging into fungus culture bags at 121 deg.C for 30min, with each bag containing 100g of wheat. Inoculating 10mL of seed solution into each bag, spreading to make the wheat uniformly infiltrate into the seed solution, and culturing in a dark constant temperature incubator at 30 ℃ for 7-10 days. Spreading the solid fermentation product in an enamel tray paved with a layer of sterile paper when the solid fermentation culture medium produces spores sufficiently, covering a layer of sterile paper, placing the enamel tray in a glass window capable of being irradiated by sunlight for airing for 5-7 days, collecting spore powder by a BFQ100 fungal spore separator after airing, and forming high spore powder, namely microbial inoculum, wherein the spore content of penicillium citrinum in the high spore powder is 1.96 multiplied by 109CFU/g。
Example 4 growth promoting action of Strain 44M-3 on six crops
The high spore powder was diluted to 2X 10 with 0.05% (v/v) Tween 807CFU/mL to obtain spore suspension for use. Sterilizing semen Maydis (Zhengdan 958), semen Tritici Aestivi (Zhongmai 18), cotton (Shandong Cotton ginning 28), fructus Cucumidis Sativi (Xinshao No. four), semen Sesami (Zhengzhi 13) and tobacco (Zhongyan 100) with 2% NaClO for 15-20min, washing with sterile distilled water for 3 times, spreading on sterile filter paper, and air drying. And (3) sterilizing soil: putting vermiculite into a nutrition pot in a ratio of 3:1, dibbling the disinfected seeds into the nutrition pot, wherein each hole has 12-18 granules, and placing the nutrition pot into a constant-temperature incubator with the temperature of 30 ℃, the illumination for 16h and the darkness of 28 ℃ for 8h for alternate light and dark cultivation. Seedlings were set at 7 plants/pot for corn and sesame, 10 plants/pot for wheat, 6 plants/pot for cotton, 7 plants/pot for cucumber, 3 plants/pot for tobacco, 3 pots for each crop treatment and control.
At the time of emergence of seedlings for 2-3d, corn, wheat, cotton, sesame and cucumber were subjected to a first root-irrigation treatment (30 mL/pot) with a 44M-3 spore suspension, and a second root-irrigation treatment was carried out according to the method of the first treatment 3d after the first treatment with 0.05% (v/v) Tween 80 treatment, and the biomass of corn and wheat was measured 7d after the second treatment, while the biomass of cotton, sesame and cucumber was measured 7d after the third treatment. The tobacco grows slowly in the early stage, the tobacco is treated for the first time 25d after seedling emergence, then treated for 1 time every 7d, the total treatment is carried out for 3 times, and the biomass is measured 7d after the 3 treatments are finished, wherein the biomass comprises the root length, the plant height, the dry weight and the wet weight of the root and the overground part. Drying at 105 deg.C for 30min, oven drying at 80 deg.C to constant weight, and weighing.
As can be seen from Table 2, spore suspensions of strain 44M-3 all had a promoting effect on the growth of corn, wheat, cotton, sesame, cucumber and tobacco roots and plants (Table 2). It contributes most to the increase of the wet weight and dry weight of the corn plant, and increases the wet weight and dry weight of the corn plant by 16.75 percent and 10.65 percent respectively; the method has the greatest contribution to the growth of the root length and the plant wet weight of wheat, and the growth rates are 4.76 percent and 4.22 percent respectively; the cotton seedling growth rate and the root wet weight increase are respectively 11.89% and 9.71%; the method has the greatest contribution to the increase of the wet weight of the sesame root, so that the wet weight of the sesame root is increased by 9.93 percent; the cucumber root wet weight and dry weight increase is maximally contributed, and the cucumber root wet weight and dry weight increase is 23.53 percent and 11.72 percent respectively; the growth contribution to the biomass of tobacco is large, and the growth rate to the dry weight of the root and the dry weight of plants of the tobacco is over 35 percent.
TABLE 2 Effect of the Strain 44M-3 on the Biomass of six crops
Example 5 preventive and therapeutic effects on blight by Penicillium citrinum 44M-3
(1) Seedling cultivation
The control is carried out by taking sesame wilt as an object. Sterilizing semen Sesami (Dillenia indica 16HJDP03) with 2% NaClO for 15-20min, washing with sterile distilled water for 3 times, spreading on sterile filter paper, and air drying. And (3) potting the sterilized soil and turfy soil according to a ratio of 3:1, dibbling the sterilized sesame seeds, placing 12 seeds in each pot, alternately culturing in a constant-temperature incubator with a temperature of 30 ℃, a light intensity of 16 h/a light intensity of 28 ℃ and a dark intensity of 8h, and fixing seedlings after 5d of seedling emergence, wherein 7 seeds in each pot are planted.
(2) Inoculation treatment
When seedlings grow to the two-leaf one-heart stage (planting for 7d), respectively carrying out root irrigation treatment on sesames of a penicillium 44M-3 group, a bacillus preparation group (biological preparation control), a carbendazim group (chemical agent control) and a blank control group by using A, B, D, D treatment liquid, wherein each 3 pots are irrigated with 30mL of roots every 3 pots, after the first treatment, 5d are irrigated with A, B, D, D treatment liquid for the second treatment, after the second treatment for 3d, the four groups of treated sesames are pulled out, roots are dipped in a pathogenic bacteria spore suspension E and then planted back, roots are irrigated with 20mL of E, after 9h, the sesames of the 44M-3 group, the bacillus preparation group, the carbendazim group and the blank control group are irrigated with A, B, C, D treatment liquid for the third treatment, and when the pathogenic bacteria are inoculated for 8d, the morbidity and the disease index are counted, and the prevention and control effect is calculated. In the first two treatments, the treatment of the carbendazim group sesame was the same as that of the blank control group, and in the third treatment, the carbendazim group was treated with a carbendazim 1000X diluent. In the method, biocontrol bacteria 44M-3 is used for preventing diseases, while carbendazim is used for treating diseases in accordance with the production.
The treatment liquids are respectively:
A. spore suspension of strain 44M-3 was prepared with 0.05% Tween 80 at a spore concentration of 1X 107CFU/mL；
B. Preparing bacillus subtilis preparation diluent with 0.05 percent of Tween 80, wherein the concentration of the bacterial suspension is 1 multiplied by 107CFU/mL (the preparation is produced by Bailide biotechnology limited company in Ningguo city, and the trade name of the preparation is 10 hundred million CFU/g bacillus subtilis wettable powder);
C. preparing 1000X diluent of carbendazim 50% wettable powder by using 0.05% Tween 80 (the carbendazim 50% wettable powder is produced by Jiangsu Lanfeng biochemical engineering Co., Ltd.);
d.0.05% tween 80;
E. spore suspension (1X 10) of the pathogenic virulent strain Fusarium oxysporum 8F27 formulated with 0.05% Tween 807CFU/mL)。
Grading standard of sesame wilt disease:
level 0: good growth without manifestation of symptoms;
level 1: leaves are slightly wilted from bottom to top, like water shortage, and can recover in the morning and evening;
and 2, stage: the diseased leaves will wilt and can not be recovered to normal;
and 3, level: the whole plant wilted and could not be recovered to normal, the vascular bundle at the stem base became yellow brown, and the whole plant died.
Disease index ═ Σ (number of diseased plants at each stage × corresponding stage value)/(investigation total number of plants × disease highest stage value) × 100 disease incidence ═ number of diseased plants/total number of treated plants × 100%
Relative control effect (disease index of control group-disease index of treatment group)/disease index of control group x 100%
Pot culture experiments show that (Table 3 and figure 2), the bacterial suspension of the strain 44M-3 can effectively prevent and treat blight in seedling stage. When 8 days of pathogenic bacteria are inoculated, the morbidity of a 44M-3 treatment group is obviously reduced, the relative control effect of the bacteria on the blight is 85.25 percent, the relative control effect is obviously superior to that of a biological preparation of bacillus subtilis, and the control effect is equivalent to that of a medicament contrast carbendazim 1000X diluent.
TABLE 3 preventive and therapeutic effects of the strain 44M-3 on blight
Example 6 growth promotion and disease prevention mechanism of Penicillium citrinum 44M-3
(1) Phosphorus dissolving capacity
As can be seen from Table 4, the bacteria can generate transparent rings on 3 culture medium plates using inorganic insoluble phosphorus as a phosphorus source, which shows that the bacteria can dissolve and utilize the three inorganic insoluble phosphorus, and the dissolving capacity of the bacteria to the three insoluble phosphorus is zinc phosphate, calcium phosphate and hydroxyapatite from strong to weak according to the ratio of the phosphorus dissolving rings to the bacterial colony diameter. The strain grows for 7 days on a culture medium without a phosphorus source, and the diameter of a bacterial colony can reach 13.98mm, which shows that the strain has strong adaptability under the condition of phosphorus deficiency.
TABLE 4 utilization of inorganic hardly soluble phosphorus by the strain 44M-3
(2) Siderophore
On a siderophin detection plate, after the strain grows to the midline 1d, a pink discoloring area is generated, the radial length is 8mm, and after the strain grows to the midline 7d, the radial length of the pink discoloring area can reach 27.5mm, which indicates that the strain has strong siderophin secretion capacity.
(3) IAA production
IAA assay was performed according to the method of Pattern and Glick (2002), IAA was dissolved in 75% ethanol to a concentration of 10 mg/mL; diluting with 75% ethanol to 5 concentrations of 0 μ g/mL (75% ethanol), 10 μ g/mL, 100 μ g/mL, 1mg/mL, and 10mg/mL, respectively, collecting IAA 10 μ L with different concentrations, adding 990 μ L PDB culture solution and 4mL LSakowski reagent, shaking, reacting at room temperature in dark for 10min, measuring absorbance with spectrophotometer (535nm), and drawing standard curve.
Culturing strain 44M-3 for 7 days to produce a large amount of conidia, eluting the conidia with 0.05% Tween 80, and adjusting the concentration of the conidia to 1 × 10 with a hemocytometer8CFU/mL, inoculating 1% into PDB culture solution, shake culturing at 28 deg.C and 180r/min for 6d, collecting bacterial liquid every day at 2-6d, centrifuging at 5500g centrifugal force for 10min, collecting supernatant 1mL, and mixing with Salkowski reagent 4mL (150mL concentrated H)2SO4250mL of distilled water, 7.5mL of 0.5M FeCl3·6H2O), standing at room temperature for 20min, measuring absorbance (535nm) with a spectrophotometer, and calculating IAA concentration according to a standard curve.
IAA standard curve:
taking the concentration of IAA as an independent variable and the light absorption value at 535nm as a dependent variable, performing regression analysis, wherein the concentration of IAA and the light absorption value have a high linear correlation relationship, namely the concentration of IAA and the light absorption value have a very high positive correlation relationship, and R20.9995 (fig. 3), and the regression curve is 0.0099x + 0.0099.
According to a standard equation and light absorption values of samples at different fermentation times, the concentration of IAA in the samples at different time points is calculated, the concentration of IAA is linearly increased when the samples are fermented for 2-4d, the concentration of IAA can reach 29.3 mu g/mL when the samples are fermented for 4d, the increase rate is gradually reduced, and the concentration of IAA tends to be stable when the samples are fermented for 6d (figure 4).
(4) Secretion of enzymes
The bacterium does not generate transparent rings when growing on a chitinase detection plate, and generates transparent rings on other three enzyme secretion detection plates (table 5), which shows that the bacterium can secrete cellulase, beta-1, 3-glucanase and protease in a dish, the cellulase secretion capacity is strongest, and beta-1, 3-glucanase and protease are respectively generated.
TABLE 5 hydrolase secretion ability of Strain 44M-3
Example 7 evaluation of biosafety of Strain 44M-3
The high spore powder is diluted to 2X 10 by 0.05 percent of Tween 807CFU/mL, spare. Sterilizing semen Maydis (Zhengdan 958), semen Tritici Aestivi (Zhongmai 18), cotton (Shandong Cotton ginning 28), fructus Cucumidis Sativi (Xinshao No. four), semen Sesami (Zhengzhi 13) and tobacco (Zhongyan 100) with 2% NaClO for 15-20min, washing with sterile distilled water for 3 times, spreading on sterile filter paper, and air drying. And (3) sterilizing soil: putting vermiculite into a nutrition pot in a ratio of 3:1, dibbling the disinfected seeds into the nutrition pot, wherein each hole has 12-18 granules, and putting the nutrition pot into a constant-temperature incubator with the temperature of 30 ℃, the illumination for 16 h/the illumination for 28 ℃ and the darkness for 8h for culture. After 5d of planting, the seedlings are fixed, and 7 corn plants and 7 sesame plants are planted in each pot, 10 wheat plants are planted in each pot, 6 cotton plants are planted in each pot, 7 cucumber plants are planted in each pot, and 3 tobacco plants are planted in each pot. And (3) when seedlings emerge for 7d, challenge inoculation is carried out, namely, the seedlings are carefully pulled out, main roots or fibrous roots are cut off by a scalpel after 1mm of the main roots or the fibrous roots are cut off, the seedlings are immersed into a 44M-3 spore suspension for root dipping treatment, after the seedlings are planted back, the seedlings are irrigated with 30mL of 44M-3 spore suspension for root irrigation treatment for the second time after 7d treatment, then the seedlings are continuously cultured for 20d, whether the crops grow normally is observed, and the treatment with 0.05% of Tween 80 is used as a control.
As a result, 20d of the culture after treatment, 6 crops of corn, wheat, cotton, sesame, cucumber and tobacco can grow normally, and compared with a control, the strain does not show any abnormal growth, which indicates that the strain does not cause diseases to the 6 crops.
Example 8 optimization of the fermentation Medium for Strain 44M-3
Earlier studies found that NaNO was removed from the formulation of the Chachi yeast extract agar medium (CYA)3And K2HPO4Then, the bacteriostatic activity of 44M-3 on the sesame pathogen fusarium oxysporum is obviously improved without adding NaNO3And K2HPO4The CYA liquid culture medium is a basic culture medium, 3 percent of glucose, sucrose, lactose, fructose, maltose, starch, glycerol, mannitol and sorbitol are respectively used for replacing a carbon source in the basic culture medium, and carbon source optimization of the culture medium is carried out by taking no carbon source as a control. After obtaining the best carbon source, 0.5 percent of yeast extract, yeast powder, beef extract, fish meal, peptone, soybean meal and NaNO are respectively added3、(NH4)2HPO4、KNO3、NH4NO3And (NH)4)2SO4The nitrogen source in the basal medium was replaced, with no nitrogen source added as a control. Each 100mL of basal medium bottle was inoculated with a 44M-3 spore suspension (2X 10) at 1%8CFU/mL), 160r/min, shake culturing at 27 ℃ for 6 days, taking the filtrate, filtering the filtrate with a 0.22-micron filter membrane for sterilization, preparing a PDA plate with 5 multiplied diluted filtrate, inoculating sesame pathogenic fusarium oxysporum in the center of the plate, taking the PDA plate added with 2mL of sterile water as a control, culturing at 28 ℃ for 7 days, measuring the colony diameter by a cross method, and calculating the bacteriostasis rate.
As can be seen from fig. 4, 44M-3 can utilize 9 carbon sources to generate bacteriostatic active substances, wherein when glucose and sucrose are used as carbon sources, the bacteriostatic activity against fusarium oxysporum is strongest, and the two have no significant difference, when sucrose is used as a carbon source, the bacteriostatic activity is more stable, and sucrose is selected as the optimal carbon source for producing the bacteriostatic active substances by fermentation. The nitrogen source optimization is carried out by taking sucrose as a carbon source, and the result shows that under the condition of no nitrogen source, the strain grows badly, the filtrate has no bacteriostatic activity, and NaNO is used in the 11 selected nitrogen sources3And KNO3When the nitrogen source is used, the fermentation filtrate has no bacteriostatic activity, 44M-3 can utilize another 9 nitrogen sources to produce bacteriostatic active substances, wherein when peptone is used as the nitrogen source, the bacteriostatic activity of the fermentation filtrate is strongest, and the result also shows that the ammonia nitrogen source is more suitable for bacterial strain fermentation to produce the bacteriostatic active substances than the nitrate nitrogen source. The nitrogen source has a greater influence on whether the strain produces the bacteriostatic active substance.
According to the optimization of carbon and nitrogen sources, the formula of the culture solution for optimally producing the antibacterial active substances is determined as follows:
30g of sucrose, 5g of peptone, 0.5g of KCl and MgSO4·7H2O 0.5g，FeSO4·7H2O0.01 g and distilled water 1000 mL.
The PDB culture solution is used for fermentation before optimization, the average bacteriostasis rate of the fermentation filtrate on 14 pathogenic fungi is 34.23%, the average bacteriostasis rate reaches 80.39% (table 6) after optimization, and compared with the method before optimization, the bacteriostasis rate of the fermentation filtrate is improved by 46.16%.
TABLE 6 bacteriostatic action of fermentation filtrate of the bacterium on 14 pathogenic fungi before and after optimization
Example 9 morphological characterization of Strain 44M-3
Uniformly coating a 44M-3 spore suspension on a PDA (personal digital assistant) plate, culturing in a dark incubator at 28 ℃ for 7 days, preparing the spore suspension by using 0.05% Tween 80, dipping the spore suspension by using a toothpick, puncturing and inoculating the spore suspension on a morphological identification culture medium, placing 4 culture plates in an incubator at 25 ℃, alternately culturing for 7 days in 16h of light and 8h of dark, simultaneously placing CYA at 5 ℃ and 37 ℃ for 7 days, observing and recording the culture characteristics of 44M-3 on four morphological culture media, measuring the colony diameter by using a cross method, and observing the microscopic morphology of the penicillium on the CYA culture medium under a microscope.
As can be seen from table 7 and fig. 5:
the strain 44M-3 grows for 7 days at 25 ℃ on a CYA culture medium, the diameter is 34.82mm, the center is yellow and flat, white convex hyphae and yellow flat hyphae alternately appear outwards, the strain is in a concentric ring pattern shape, no exudate and no soluble pigment exist, and the reverse side is yellow and brown alternate concentric ring patterns.
The strain 44M-3 grows for 7d at 25 ℃ on an MEA culture medium, the diameter is 23.04mm, the center is white and convex, other parts are flat and velvet, the number of conidia is large, the conidia surface is blue-green, no exudate exists, light brown soluble pigment is produced, and the reverse side is light yellow.
The strain 44M-3 grows for 7d at 25 ℃ on a CA culture medium, the diameter is 18.45mm, the center of a bacterial colony is raised, a small amount of conidia are generated, the spore surface is greenish, the reverse surface is nearly transparent, no exudate exists, and the soluble pigment is lack.
The strain 44M-3 grows for 7d at 5 ℃ on a CYA culture medium, the diameter is 4.63mm, the center is convex, the front surface and the back surface are both white, and no spore is produced.
The strain 44M-3 grows for 7d at 37 ℃ on a CYA culture medium, the diameter is 4.36mm, the whole colony is white and convex, the reverse side is reddish brown, and no spore is produced.
The strain 44M-3 is subjected to three-round-robin-generation, and conidia are spherical, nearly spherical to elliptical.
TABLE 744M-3 growth rates on four morphological media
Example 10 molecular biological characterization of Strain 44M-3
Extracting hyphal genome DNA by a CTAB (cetyl trimethyl ammonium bromide) method, taking the genome DNA as a template, and respectively extracting the hyphal genome DNA by using a specific primer Bt2a of a beta-tubulin sequence: 5'-GGTAACCAAATCGGTGCTGCTTTC-3' and Bt2 b: 5'-ACCCTCAGTGTAGTGACCCTTGGC-3', and primers specific for the Calmodulin sequence CMD 5: 5 '-CCGAGTACAAGGARGCCTTC-3' and CMD 6: 5 '-CCGATRGAGGTCATRACGTGG-3' was subjected to PCR amplification using 2 XTaq Master Mix 12.5. mu.L, 1. mu.L each of the forward primer (10. mu. mol/L) and the reverse primer (10. mu. mol/L), 1. mu.L of DNA template, and the complement of ddH2O to 25. mu.L.
PCR amplification conditions were 94 ℃ for 5 min; at 94 ℃ for 45s, at 55 ℃ for 45s, at 72 ℃ for 1min, for 35 cycles; 7min at 72 ℃. The PCR product was electrophoresed on 2% agarose gel, and the product was recovered using a gel recovery kit. Connecting the recovered product to PMD-19T-vector, transforming competent (DH5 alpha) cells, shaking the competent cells at 37 ℃ and 160r/min until the cells are turbid, carrying out PCR amplification on bacterial liquid, detecting the PCR product by using 1% agarose gel electrophoresis, selecting the bacterial liquid with the electrophoresis result positive, sending the bacterial liquid to Shanghai Biotech limited company for sequencing, and sending three cloned bacterial liquids to each sample.
Performing Blast homologous sequence retrieval on a sequence returned by a sequencing company in GenBank, selecting a strain with higher homology as a reference object, performing sequence editing and multi-sequence comparison by adopting Bioedit 5.0 and MEGA 7.0 software, and constructing a phylogenetic tree by using an adjacent method.
44M-3 beta-tubulin and Calmodulin were subjected to sequencing by a worker, and a 465bp beta-tubulin sequence and a 497bp Calmodulin sequence were respectively returned, and Blast results on NCBI found that the 44M-3 beta-tubulin and Calmodulin sequences have high similarity with Penicillium aurantium, Penicillium virdicatum and Penicillium freii. After Blast on NCBI, the β -tubulin sequence of 44M-3 has 98.92%, 98.28% and 98.24% similarity to P.aurantigresum, P.viridiatum and P.freii, respectively, on the phylogenetic tree of β -tubulin, 44M-3 and P.aurantigresum clustered in the same branch, and bootstrap-verified support rate reached 96 (FIG. 6A).
The similarity of the Calmodulin sequence to P.aurantigresum, P.viridiatum and P.freii after Blast on NCBI is 99.80%, 98.59% and 98.59%, respectively, 44M-3 also gathers in the same branch with P.aurantigresum on the phylogenetic tree of Calmodulin, and the bootstrap validation support rate reaches 96 (FIG. 6B). The bootstrap verification rate may be a subspecies or a variety from 66 to 95, and the same species can be judged above 95. According to the evolutionary development analysis of the beta-tubulin and Calmodulin sequences and the morphological characteristics on the identification culture medium, 44M-3 is identified as Penicillium citrinum P. 44M-3 beta-tubulin and Calmodulin sequences have been uploaded to the NCBI database under sequence accession numbers MN325973 and MN325971, respectively.
Claims (8)
1. The Penicillium citrinum is characterized in that the Penicillium citrinum is Penicillium citrinum (Penicillium aurantiogenum) 44M-3, is preserved in China center for type culture collection, and has the preservation address: wuhan university collection center, accession number: CCTCC NO: m2019768, date of deposit: 09 and 30 days 2019.
2. A method for preparing a high sporopollen microbial inoculum by using penicillium citrinum strains is characterized by comprising the following steps:
inoculating penicillium citrinum to a PDA culture medium, alternately culturing in a constant-temperature incubator with 25 ℃ and 8h of illumination/16 h of darkness for 5-7d in a light-dark manner, eluting spores by using 0.05% (v/v) Tween 80 sterile water to form a spore suspension, subpackaging PDB culture solution into 300mL triangular bottles, subpackaging 200mL each bottle, inoculating 5mL of the spore suspension to each bottle, culturing at 180r/min at 28 ℃ for 4d in a shaking way, and taking the spore suspension as a seed solution for solid fermentation culture for later use;
weighing 1500g of wheat, soaking in distilled water for 8h, taking out, draining, and packaging into fungus culture bags at 121 deg.C for 30min, each bag containing 100g of wheat; inoculating 10mL of seed solution into each bag, spreading to make the wheat uniformly infiltrate the seed solution, and culturing in a dark constant-temperature incubator at 28 ℃ for 7-10 days;
spreading the solid fermentation product in an enamel tray paved with a layer of sterile paper when the solid fermentation culture medium produces spores sufficiently, covering a layer of sterile paper, placing the enamel tray in a glass window capable of being irradiated by sunlight for airing for 5-7 days, collecting spore powder by using a BFQ100 fungal spore separator after airing, and forming a high-spore-powder microbial inoculum, wherein the spore content of penicillium citrinum in the high-spore-powder microbial inoculum is 1.96 multiplied by 109CFU/g。
The PDA culture medium: peeling 200g of potatoes, cutting into blocks, putting the cut potatoes into distilled water, boiling for 10min, filtering through 6 layers of gauze, adding 20g of glucose and 15-20 g of agar into filtrate, adding distilled water to a constant volume of 1000mL, and sterilizing for 30min at 121 ℃;
the PDB culture solution is as follows: peeling potato 200g, cutting into pieces, putting into distilled water, boiling for 10min, filtering with 6 layers of gauze, adding glucose 20g into the filtrate, adding distilled water to reach a constant volume of 1000mL, and sterilizing at 121 ℃ for 30 min.
3. The method of using the high spore powder microbial inoculum according to claim 2, wherein the high spore powder is re-suspended with sterile water of 0.05% (v/v) tween 80, the spore concentration is adjusted by counting with a blood counting plate, and the roots of plants are treated by dipping or irrigating the roots with spore suspension liquid of different concentrations.
4. A culture solution for producing bacteriostatic active substances by penicillium citrinum strains is characterized in that the formula of the culture solution is as follows:
30g of sucrose, 5g of peptone, 0.5g of KCl and MgSO4·7H2O 0.5g，FeSO4·7H2O0.01 g and distilled water 1000 mL.
5. Use of penicillium citrinum according to claim 1 for inhibiting the growth of pathogenic bacteria of wheat, tobacco, sesame, rehmannia and osmanthus diseases.
6. Use of penicillium citrinum as claimed in claim 1 for promoting the growth of corn, wheat, cotton, sesame, cucumber and tobacco.
7. Use of penicillium citrinum as defined in claim 1 for the prevention and treatment of sesame wilt.
8. The method for preventing and treating sesame wilt disease by penicillium citrinum as claimed in claim 7, which is characterized in that: spore suspension of Penicillium citrinum strain with 0.05% (v/v) Tween 80 at spore concentration of 1 × 107CFU/mL; and (3) irrigating roots of the sesames by using the spore suspension at the seedling stage of the sesames.
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