CN109022308B - Pseudomonas putida BC10 and application thereof in preventing and treating crop bacterial soft rot - Google Patents
Pseudomonas putida BC10 and application thereof in preventing and treating crop bacterial soft rot Download PDFInfo
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Abstract
The invention discloses a pseudomonas putida strainPseudomonas putida) BC10 and application thereof in preventing and treating bacterial soft rot of crops. The strain is preserved in Guangdong province microorganism culture collection center in 2018, 7 months and 10 days, and the preservation number is GDMCC NO. 60411. The strain has obvious inhibition effect on various bacterial disease bacteria, especially on rice bacterial basal blight bacteria, banana bacterial soft rot bacteria and kaffir lily soft rot bacteria, can reduce the occurrence of crop bacterial soft rot, has good prevention and control effect in a potting test, has no pathogenicity in the strain BC10, and shows that the strain can be used for developing biological pesticides for crop bacterial diseases.
Description
Technical Field
The invention belongs to the technical field of plant disease biocontrol. In particular to pseudomonas putida BC10 and application thereof in preventing and treating bacterial soft rot of crops.
Background
Dieckea bacteria (D) (Dickeya) Is one of ten important plant pathogenic bacteria in the world, is listed as No. 302 quarantine harmful organisms in China, and can cause serious bacterial soft rot of crops and flowers. Wherein the content of the first and second substances,D. dadantii、D. zeaeandD. solanioften cause serious crop diseases, especiallyD. zeaeInduced bacterial base of riceRot and bacterial soft rot of banana, andD. solanithe bacterial soft rot of potato caused by the method has a tendency of continuously expanding and aggravating in recent years.
Dieckea zeae (D)D. zeae) The host range of (1) is wide, besides monocotyledons, dicotyledons can be infected at the same time, and the host range of (1) comprises rice, corn, sugarcane, banana, brachiaria, iris, blackberry lily, hemerocallis, pennisetum, pineapple, potato, tobacco, Chinese cabbage, carrot, peanut, impatiens balsamina, chrysanthemum and the like. In 2009, massive outbreaks of banana soft rot in Guangdong province resulted in about 6000 hectares of bananas affected between 2010 and 2012. According to investigation, the disease rate of the disease is increased year by year in the Guangdong, and the field soft rot and the blight are seriously infected in a complex way. At present, the disease has spread to Fujianzhang state, eastern Hainan county, Ledongli autonomous county, Lingao county, Chengmei county, Yunnan Xishuangbanna, Guangxi Nanning and other places.
The pathogenic bacteria have high propagation speed, various propagation ways, long survival time in soil, obvious differentiation of a bacterial system, high mutation speed and complex pathogenic mechanism, so the prevention and the treatment are difficult. The existing prevention and control methods mainly comprise the following steps: (1) planting disease-resistant varieties. It is the most economic and effective method for preventing and controlling banana bacterial soft rot. However, with the increase of the planting time of the disease-resistant variety, the pathogenic bacteria may generate new physiological races, thereby turning the disease-resistant variety into a disease-susceptible variety. (2) Chemical agents are used. At present, the medicines for preventing and treating bacterial diseases including soft rot are few in varieties, and mainly comprise copper preparations, antibiotic bactericides and the like. However, chemical agents have certain toxicity to human and livestock, and can cause problems of drug resistance of germs, water resource pollution and the like.
Therefore, the search for suitable biological control methods is a development direction and an effective way to control bacterial soft rot.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the existing bacterial soft rot control technology, provides a biocontrol bacterium pseudomonas putida BC10 with better inhibiting effect on various crop bacterial soft rot germs, provides a new way for replacing a chemically synthesized bactericide with microorganisms, and can be developed and utilized as a biological pesticide.
The invention also aims to provide application of the pseudomonas putida BC10 in preventing and treating bacterial soft rot of crops.
Another object of the invention is to provide a biological agent for preventing and treating bacterial soft rot of crops.
Another object of the present invention is to provide a method for controlling bacterial soft rot of crops.
The above purpose of the invention is realized by the following technical scheme:
the invention obtains a strain of pseudomonas putida by screening and identifyingPseudomonas putida) BC10, and is preserved in Guangdong province microorganism culture collection center in 2018, 7 and 10 months, with the preservation number of GDMCC NO.60411 and the preservation address of No. 59, 5 th of Dazhou 100, Jieli, Guangzhou city.
BC10 has round colony on LB culture medium, milky yellow color, smooth and regular edge, slightly raised middle part, opacity, gram-negative color, capacity of liquefying gelatin, producing acid and catalase, no amylase, fructose, small amount of trehalose and no galactose, mannitol, glucose and sucrose.
Researches show that the strain BC10 can form an obvious inhibition zone on a flat plate containing bacterial liquids of rice bacterial basic rot EC1, banana bacterial soft rot MS2 and MS3, does not influence the growth of the banana seedlings in a greenhouse potting test, and can effectively inhibit the infection of MS2 on the banana seedlings. In addition, the growth inhibition effect of the strain BC10 on a plurality of plant pathogenic bacteria including solanaceae ralstonia solanacearum, rice leaf blight, rice soft rot, peach rot and a plurality of plant pathogenic fungi including pepper anthracnose, collodion anthracnose, banana fusarium wilt, rice sheath blight, peronophythora litchii and sugarcane smut is not obvious, which shows that the strain BC10 can be used for developing biological pesticides special for crop bacterial soft rot. The discovery of the strain is beneficial to relieving the abuse problem of chemical agents, and provides resources for preventing and treating the bacterial soft rot of plants by using a biological control method.
Therefore, the application of the pseudomonas putida BC10 in preventing and controlling the bacterial soft rot of crops also belongs to the protection scope of the invention.
Preferably, the bacterial soft rot disease comprisesDickeyaOrErwiniaCausing bacterial soft rot.
More preferably, the bacterial soft rot includes, but is not limited to, bacterial soft rot on banana, clivia, radish, carrot, chinese cabbage, potato or rice bacterial basal rot.
The invention also claims a biological preparation for preventing and controlling the bacterial soft rot of crops, which comprises the pseudomonas putida BC 10.
Preferably, the concentration of the pseudomonas putida BC10 is OD600=1.0～1.5。
More preferably, the concentration of Pseudomonas putida BC10 is OD600=1.3。
Preferably, the biological agent is prepared from a bacterial liquid and/or a fermentation liquid of pseudomonas putida BC 10.
The invention also claims a method for preventing and controlling the bacterial soft rot of crops, which is realized by treating soil or plant materials with the pseudomonas putida BC10 or the biological agent.
Preferably, the treatment is a seeding treatment.
More preferably, the inoculation treatment is an injection inoculation method or a puncture inoculation method.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a pseudomonas putida BC10, which has obvious inhibition effect on various bacterial disease bacteria, especially on rice bacterial basal blight bacteria, banana bacterial soft rot bacteria and kaffir lily soft rot bacteria, can reduce the occurrence of crop bacterial soft rot, has good prevention and control effect in a pot experiment, and has no pathogenicity in the strain BC10, which indicates that the strain can be used for developing biological pesticides aiming at crop bacterial diseases and can be used for biologically preventing and controlling the crop bacterial soft rot.
Drawings
FIG. 1 is a clustering diagram of strain BC10 based on the 16S rDNA gene sequence.
FIG. 2 shows that strain BC10 is based ongyrBCluster map of gene sequences.
FIG. 3 shows that strain BC10 is based onrpoBCluster map of gene sequences.
FIG. 4 shows that strain BC10 is based onrpoDCluster map of gene sequences.
FIG. 5 shows the determination of the bacteriostasis spectrum of biocontrol bacterium BC10 on pathogenic bacteria of important crops.
FIG. 6 is the determination of the bacteriostasis spectrum of the biocontrol bacterium BC10 on the pathogenic fungi of important crops.
FIG. 7 shows that strain BC10 significantly reduced the occurrence of bacterial soft rot disease in dicotyledonous host plants.
FIG. 8 shows that the strain BC10 remarkably reduces the occurrence of banana bacterial soft rot disease symptoms.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 isolation and screening of Pseudomonas putida BC10
1. Strain isolation
(1) Collecting samples: soil samples with soil surface of 5 cm below are dug in the cabbage rhizosphere soil in village under the great mountains of Committee in the country of Kyoho of Kwangsi Hepu Qu-zhang, and the cabbage rhizosphere soil is bagged and stored and taken back to a laboratory for strain separation.
(2) Strain separation: the soil sample is subjected to strain separation by adopting a dilution coating plate method. Specifically, the collected soil sample is fully and uniformly mixed by hand, 4.0 g is weighed, soaked and uniformly shaken in 20 mL sterile water, and then shake-cultured for 20 min at 28 ℃ and 180 rpm for gradient dilution. Taking the dilution factor as 105～109The diluted solution of 100 mu L is coated on an LB flat plate, and is sealed after being dried at room temperature, and is inverted and cultured in an incubator at 28 ℃ for 24 hours. Picking LBSingle colonies growing on the flat plate are marked and placed in a 2 mL centrifuge tube containing 500 muL of liquid LB, and shaking culture is carried out on a shaking table at 180 rpm and 28 ℃ overnight for later use.
2. Strain screening
Activating banana bacterial soft rot pathogen MS2 on an LB plate, picking a single colony in a 50mL centrifuge tube containing 15 mL LB culture solution, shaking and culturing at 28 ℃ and 180 rpm by shaking overnight for standby. Pouring 15 mL of melted solid LB culture medium in a square culture dish of 10 x 10 cm, drying, pouring 15 mL of agarose (cooling to 50-60 ℃ and adding 150 muL of MS2 bacterial liquid respectively) with the concentration of 1% on the square culture dish paved with LB, and drying. And punching by using a puncher with the diameter of 5 mm, after the culture medium is picked up by the sterilized toothpick, injecting 20 mu L of overnight-cultured bacterial liquid to be detected into the hole, drying and sealing. And (3) placing the plate in an incubator at 28 ℃, culturing for 16-18 h, and observing whether a transparent bacteriostatic zone exists. Obtaining a biocontrol strain with the best bacteriostatic effect, and naming the biocontrol strain as BC 10.
Example 2 identification of Pseudomonas putida BC10
1. Morphological and physiological biochemical identification
BC10 has round colony on LB culture medium, milky yellow color, smooth and regular edge, slightly raised middle part, opacity, gram-negative color, capacity of liquefying gelatin, producing acid and catalase, no amylase, fructose, small amount of trehalose and no galactose, mannitol, glucose and sucrose.
2. Molecular identification
To clarify the classification status of the strain BC10 obtained in example 1, the BC10 strain was identified by a method of multilocus sequence analysis (MLSA) combined with Phylogenetic tree analysis (Phylogenetic analysis).
The 16S rDNA sequence analysis is a molecular biological identification means for identifying bacteria, and the invention combines the conserved housekeeping gene by analyzing the 16S rDNA sequencegyrB、rpoBAndrpoDand (3) constructing a phylogenetic tree by applying MEGA 6.0 software and adopting a Neighbor-join method, and comprehensively analyzing the evolutionary relationship of BC 10.
The results show that: 16S rDNA sequence of BC10 andPseudomonas putidaL1-5、PC2、P. entomophilaL48、P. mosseliithe homology of PtA1 reaches 99.80%, 99.46%, 99.46% and 99.40%, respectively, and phylogenetic relationship analysis shows that the PtA1 is in the same branch with the strains of the three species (as shown in figure 1);gyrBgene sequences andP. putidaL1-5、P. entomophilaL48、P. mosseliithe homology of PtA1 reaches 98.93%, 93.58% and 93.03% respectively;rpoBgene sequences andP. vranovensis15D11、P. putidaPC2、P. alkylphenolicaKL28、P. entomophilaL48、P. mosseliithe homologies of SJ10 and PtA1 reach 95.44%, 95.04%, 94.87%, 94.14%, 94.06% and 93.90% respectively;rpoDgene sequences andP. entomophilaL48、P. putidaCFBP5934、P. plecoglossicidaXSDHY、P. mosseliithe homologies of SJ10 and PtA1 reach 92.84%, 92.21%, 89.77%, 89.11% and 89.00%, respectively. Combining the phylogenetic relationship of the 4 genes, determining BC10 and BCP. putidaPC2 has the closest genetic relationship (see FIGS. 2-4).
In conclusion, except for the differences between gelatin liquefaction in physiology and biochemistry and Pseudomonas putida reported, the other components are consistent with the characteristics of Pseudomonas putida. Therefore, the biocontrol bacterium BC10 was identified as Pseudomonas putida. And the strain is preserved in Guangdong province microorganism culture collection center in 2018, 7 and 10 days, the preservation number is GDMCC NO.60411, and the preservation address is No. 59 building 5 building of the Reliao Zhou 100 Dazhong college in Guangzhou city.
Example 3 determination of the Pseudomonas putida BC10 bacterial inhibition Spectrum
1. Determination of the inhibition Spectrum
In order to better study the biocontrol potential of the strain BC10, the bacterial inhibition spectrum of the strain is studied. The specific operation is as follows:
(1) it was investigated whether BC10 has the ability to inhibit the growth of other pathogenic bacteria. The same method as that for screening the strain in example 1 is used, the pathogenic bacteria MS2 is replaced by the same amount of rice basal rot EC1, banana bacterial soft rot MS2 and MS3, ralstonia solanacearum EP1 (15 mL LB of the first layer is replaced by 15 mL of TTC), rice bacterial leaf blight Xoo, rice soft rot SC7 and peach rot PP1, and the rest steps are unchanged.
(2) Whether BC10 has the ability to inhibit the growth of pathogenic fungi was explored. The fungi to be tested include colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rhizoctonia solani, peronospora litchii and ustilago. Wherein the smut pathogen adopts a plate confronting method. 20 mL of PDA was placed on a 13X 13 square petri dish and cut into rectangular strips with a width of 0.5 cm and a length of 6 cm, each block being 0.5 cm apart, after the plates were dried. And (3) putting 1 mu L of BC10 bacterial liquid cultured overnight at one end of the PDA strip, and uniformly putting the smut "+" basidiospore and "-" basidiospore and the mixed liquid of two basidiospores in equal amount in the middle. And (3) after the bacterial liquid is dried, placing the bacterial liquid in an incubator at 28 ℃ for inverted culture for 1-2 days, observing the growth condition of the smut, and recording the test result after white villous hyphae are formed on a control plate.
Punching holes with a 0.5 cm puncher after the rest fungi are recovered, inversely placing the fungus cakes in the center of a PDA (personal digital assistant) plate, dripping 2 muL of the BC10 and control escherichia coli DH5 α bacterial liquid cultured overnight at the same distance from the fungus cakes, sealing after the bacterial liquid is dried, just placing the bacterial liquid in an incubator at 28 ℃ for culturing for 3-4 days, observing the growth condition of hyphae and recording the experimental result.
2. Results of the experiment
The strain BC10 has strong bacteriostatic action on rice basal rot fungi EC1, banana bacterial soft rot fungi MS2 and MS3, and has strong bacteriostatic action on solanaceae ralstonia solanacearum EP1, rice bacterial blight Xoo and rice soft rot fungiPantoea ananatisSC7, peach rot pathogenP. ananatisPP1 has undesirable bacteriostatic effect (see FIG. 5); the bacteriostatic effects on colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rice sheath blight, rice blast and smut are weak (fig. 6), which shows that BC10 can be used for developing a biocontrol agent specially aiming at crop bacterial soft rot, and has strong specificity.
Example 4 determination of the Effect of Pseudomonas putida BC10 on bacterial Soft rot
1. Activating banana bacterial soft rot MS2 and biocontrol bacteria BC10 on an LB solid plate, selecting a single colony to 50mL containing 10 mL of LBPlacing in a centrifuge tube, shaking at 28 deg.C and 180 rpm for overnight culture, and collecting 1 mL OD600The BC10 bacterial liquid (about 1.3) was mixed with an equal amount of MS2 bacterial liquid. The MS2 bacterial liquid is added with 1 mL LB bacterial liquid to be mixed as positive control, and LB culture medium is used as negative control.
Radish, carrot, Chinese cabbage, potato and banana seedlings are respectively inoculated in different treatments. Each treatment was 3 replicates. Selecting 12 pink banana seedlings with similar growth vigor, respectively taking 200 mu L of LB, MS2+ LB, BC10+ LB and MS2+ BC10 mixed liquid by using a1 mL injector, injecting the mixed liquid into pseudostems of the pink bananas, repeating the treatment for 3 times, observing the growth condition of the pink banana seedlings every day, and recording the growth condition.
2. Experimental results show that the strain BC10 can obviously reduce the incidence rate of bacterial soft rot on radish, carrot, Chinese cabbage, potato (shown in figure 7) and banana (shown in figure 8).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The Pseudomonas putida (Pseudomonas putida) BC10 is preserved in Guangdong province microorganism culture collection center in 2018, 7 months and 10 days, and the preservation number is GDMCC NO. 60411.
2. The application of the pseudomonas putida BC10 as claimed in claim 1 in preventing and treating the bacterial basal rot of crops caused by the rice basal rot fungus EC 1.
3. The use of pseudomonas putida BC10 as claimed in claim 1 for controlling bacterial soft rot of crops caused by banana bacterial soft rot pathogen MS2 and MS 3.
4. The use according to claim 3, wherein the crops are radish, carrot, Chinese cabbage, potato and banana.
5. A biological agent for controlling bacterial soft rot of crops, comprising pseudomonas putida BC10 according to claim 1.
6. The biological agent as claimed in claim 5, wherein the concentration of Pseudomonas putida BC10 is OD600 ═ 1.0-1.5.
7. The biological agent according to claim 5, wherein the biological agent is prepared from a bacterial liquid and/or a fermentation liquid of Pseudomonas putida BC 10.
8. A method for controlling bacterial soft rot of crops, characterized in that soil or plant material is treated with pseudomonas putida BC10 according to claim 1 or a biological agent according to any one of claims 5 to 7.
9. The method of claim 8, wherein the treatment is a seeding treatment.
10. The method of claim 9, wherein the vaccination treatment is an injection vaccination or a puncture vaccination.
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| CN111996232A (en) * | 2020-08-21 | 2020-11-27 | 中国农业科学院郑州果树研究所 | Method for screening peach soft rot medicament based on pathogen separation identification and microplate method |
| CN112522136A (en) * | 2020-11-19 | 2021-03-19 | 贵州省烟草科学研究院 | Pseudomonas and application thereof in controlling bacterial wilt of solanaceae crops |
| CN113416731B (en) * | 2021-07-08 | 2022-11-11 | 华南农业大学 | Reporting system for screening quenching bacteria and inhibitors of VFM (vacuum fast transient response) quorum sensing signals and construction method of reporting system |
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| EP1241247A1 (en) * | 2001-03-13 | 2002-09-18 | C.C.H. S.A. | Isolated bacteria for the protection of plants against phytopathogenic fungi and bacteria |
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