CN108624526B - Pseudomonas fluorescens SC3 and application thereof in preventing and treating crop bacterial soft rot - Google Patents
Pseudomonas fluorescens SC3 and application thereof in preventing and treating crop bacterial soft rot Download PDFInfo
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Abstract
The invention discloses pseudomonas fluorescens (A)Pseudomonas fluorescens) SC3 and its application in preventing and treating bacterial soft rot of crop. The strain of pseudomonas fluorescens SC3 has been deposited in Guangdong provincial microorganism culture collection at 2018, 4 and 30 months, and the deposit number is GDMCC number 60367. The strain SC3 has obvious inhibition effect on various bacterial disease bacteria, especially has good inhibition effect on crop bacterial soft rot, ralstonia solanacearum and xanthomonas oryzae, reduces the occurrence of crop bacterial soft rot, and has good prevention and control effect in pot experiment. Moreover, the bacterial strain SC3 has no pathogenicity, can be used for developing biological pesticides aiming at crop bacterial diseases, and provides a new idea and a new idea for biologically preventing and treating crop bacterial soft rot.
Description
Technical Field
The invention belongs to the technical field of plant disease biocontrol. More particularly, relates to pseudomonas fluorescens SC3 and application thereof in preventing and treating bacterial soft rot of crops.
Background
Bacterial diseases are plant diseases caused by bacterial pathogen infection, such as soft rot, bacterial wilt and the like, and cause serious economic loss. The symptoms of bacterial diseases are manifested as wilting, rotting, withering and the like, and bacterial mucus overflows from the disease parts when wet weather occurs in the later period of disease, so that the disease has obvious foul smell and is characterized by bacterial diseases.
Such as Dieckea zeae (D.), (Dickeya zeae) The host range of the bacterium is wide, and the bacterium can infect monocotyledon and can invade monocotyledon simultaneouslyDicotyledonous plants are selected from rice, corn, sugarcane, banana, brachiaria, iris, blackberry lily, hemerocallis, pennisetum, pineapple, potato, tobacco, Chinese cabbage, carrot, peanut, balsam, chrysanthemum, etc. Has been reported to be composed ofD. zeaeThe caused serious plant diseases comprise rice bacterial basal rot and banana bacterial soft rot, and the caused diseases have latent infection phenomena, so that a large amount of serious yield loss of crops is caused.
The pathogenic bacteria have fast propagation speed and various propagation ways, can survive in soil for a long time, and has obvious differentiation of bacterial systems, fast variation 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: the method is the most economical and effective method for preventing and treating the bacterial soft rot of rice and bananas. Researches show that plantain, Brazil banana and emperor banana have certain resistance to 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) Using chemical agents: 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. The research results of Chen Yuanfeng and the like show that chlorine dioxide (ClO) higher than 5 mg/L is used2) The water body disinfection can effectively prevent the bacterial soft rot from spreading through water source, and more than 1500 mg/L of ClO is used2The aim of eliminating pathogens can be achieved by disinfecting the diseased banana pit soil. In addition, research indicates that the hydromycin and the chlorotoxin have the effect of preventing and treating the bacterial soft rot disease of the bananas, but field tests are not carried out. 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 diseases.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing bacterial disease control technology, provides a biocontrol bacterium with better inhibiting effect on various crop bacterial disease bacteria, provides a new development resource for replacing a chemically synthesized bactericide with microorganisms, and can be developed and utilized as a biological pesticide.
The invention aims to provide pseudomonas fluorescens SC 3.
The invention also aims to provide application of the pseudomonas fluorescens SC3 in preventing and treating crop bacterial diseases.
The above purpose of the invention is realized by the following technical scheme:
the invention obtains a strain of pseudomonas fluorescens by screening and identifyingPseudomonas fluorescens) SC3, and is preserved in Guangdong province microorganism culture collection center in 2018, 4 and 30 months, with the preservation number of GDMCC number 60367, and the preservation address of No. 59, No. 5, of the Michelia Tokyo 100, Guangzhou city.
Researches show that the strain SC3 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 rice and banana seedlings in greenhouse potting experiments, and can effectively inhibit the infection of the EC1 on the rice and the MS2 on the banana seedlings. In addition, SC3 is found to have strong bacteriostatic action on a plurality of plant pathogenic bacteria including solanaceae ralstonia solanacearum EP1, Brassica napus xanthomonas sensation Xcc and Paddy rice bacterial blight Xoo. But the growth inhibition effect on a plurality of plant pathogenic fungi including colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rhizoctonia solani, pyricularia oryzae and sugarcane smut is not obvious. The bacterial strain SC3 of the invention can be used for developing biological pesticides aiming at crop bacterial diseases. The discovery of the strain is beneficial to reducing the abuse problem of chemical agents, and provides resources for preventing and treating plant bacterial diseases by using a biological control method.
Therefore, the application of the pseudomonas fluorescens SC3 in the aspect of controlling crop bacterial diseases also falls within the protection scope of the invention.
Preferably, the crop bacterial diseases include, but are not limited to, bacterial diseases caused byDickeya zeaeOrErwiniaCausing bacterial diseases.
Preferably, the bacterial diseases comprise bacterial basal rot of rice, bacterial soft rot, solanaceae bacterial wilt, cruciferae black rot or rice bacterial leaf blight and the like.
Wherein the bacterial soft rot disease comprises bacterial soft rot disease of banana, kava, kaffir lily, radish, carrot, Chinese cabbage, potato, etc.
The solanaceae bacterial wilt is composed ofRalstonia solanacearumAnd (4) causing. The cruciferae black rot is prepared fromXanthomonas campetrispv.campetrisAnd (4) causing. The bacterial leaf blight of rice is composed ofXanthomonas oryzaepv.oryzaeAnd (4) causing.
In addition, a biocontrol agent for crop bacterial diseases, which contains the pseudomonas fluorescens SC3, also falls within the protection scope of the present invention.
Preferably, the concentration of Pseudomonas fluorescens SC3 in the biocontrol preparation is OD6001.0～1.5。
As an alternative embodiment, the invention also provides a method for controlling crop bacterial diseases, wherein the biocontrol agent is inoculated to plant materials.
Preferably, the inoculation can be by injection inoculation.
Preferably, the inoculation can be a stab inoculation.
The invention has the following beneficial effects:
the pseudomonas fluorescens SC3 provided by the invention has a remarkable inhibition effect on various bacterial disease bacteria, especially has a good inhibition effect on crop bacterial soft rot, pseudomonas solanacearum and xanthomonas flavipes, reduces the occurrence of the crop bacterial soft rot, and has a good prevention and control effect in a potting test.
The research of the invention shows that the strain SC3 can form an antibacterial ring on a flat plate containing EC1, MS2, MS3, EP1, Xcc and Xoo bacterial liquid; in an inoculation test, SC3 has no pathogenicity on radishes, carrots, cabbages, potatoes, rice and plantain, but can well inhibit the infection of pathogenic bacteria on the radishes, the carrots, the cabbages, the potatoes, rice seedlings and the plantain seedlings and reduce the incidence of bacterial soft rot.
Moreover, the bacterial strain SC3 has no pathogenicity, can be used for developing biological pesticides aiming at crop bacterial diseases, and provides a new idea and a new idea for biologically preventing and treating crop bacterial soft rot.
Drawings
FIG. 1 is a clustering plot of strain SC3 based on the 16S rDNA gene sequence.
FIG. 2 shows that the strain SC3 is based ongyrBCluster map of gene sequences.
FIG. 3 shows that the strain SC3 is based onrpoBCluster map of gene sequences.
FIG. 4 shows that the strain SC3 is based onrpoDCluster map of gene sequences.
FIG. 5 shows the determination of the bacteriostasis spectrum of the biocontrol bacterium SC3 on pathogenic bacteria of important crops.
FIG. 6 shows the determination of the bacteriostasis spectrum of the biocontrol bacterium SC3 for important crop pathogenic fungi.
FIG. 7 shows that the strain SC3 significantly reduces the occurrence of bacterial soft rot disease symptoms of dicotyledonous host plants.
FIG. 8 shows that the strain SC3 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 SC3
1. Strain isolation
Collecting samples: in the rice field of Sichuan agricultural university, a plant with rotten base of rice seedlings is pulled out, and the plant and the soil sample of the root of the rice are bagged, stored and taken back to a laboratory for strain separation.
Strain separation: the soil sample is subjected to strain separation by adopting a dilution coating plate method. Specifically, the collected soil sample is thoroughly mixed by hand, and then 4.0 g of the mixture is weighed and used in 20 mL of the mixtureSoaking the strain in water, shaking at 28 deg.C and 180 rpm, and performing shake culture for 20 min 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 single colonies growing on the LB flat plate, labeling, placing in a 2 mL centrifuge tube containing 500 muL liquid LB, shaking and culturing at 28 ℃ and 180 rpm by shaking overnight for later use.
2. Strain screening: activating banana bacterial soft rot pathogen MS2 on an LB plate, picking a single colony in a 50 mL 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 with the concentration of 1% (cooling to 50-60 ℃ and adding 150 muL of MS2 bacterial liquid respectively) 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. The plate is placed in an incubator at 28 ℃ and cultured for 16-18 h, and whether a transparent bacteriostatic zone exists or not is observed. Obtaining a biocontrol strain with the best bacteriostatic effect, and naming the biocontrol strain as SC 3.
Example 2 identification of Pseudomonas SC3
1. Morphological identification
Strain SC3 is gram negative; the bacterial colony on the LB nutrient medium is light yellow and round, the middle of the bacterial colony is slightly convex, the surface of the bacterial colony is smooth, and the edge of the bacterial colony is neat; the liquid culture medium is diffusible turbid, gelatin is liquefied, and acid, gas, oxidase and catalase are produced without producing amylase.
2. Molecular identification
To clarify the taxonomic status of the strain SC3 obtained in example 1, we identified the SC3 strain by a method of Multilocus sequence analysis (MLSA) in combination 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、rpoBAndrpoDthe sequence, a phylogenetic tree is constructed by applying MEGA 5.0 software and adopting a Neighbor-join method, the evolutionary relationship of SC3 is comprehensively analyzed, and researches show that the 16S rDNA sequence of SC3 and pseudomonas arePseudomonas fluorescensThe homology of 48D1 and Pfo-1 strains reaches the highest 99%, and the evolutionary coefficients are consistent with that of the Pfo-1 strainsP. fluorescens48D1 (FIG. 1) was most closely matched by analysisgyrB、rpoBAndrpoDgene sequence discovery, SC3 andP. fluorescens48D1 is closest to LMG14673 (see fig. 2-4).
In summary, the biocontrol bacteria were identified as Pseudomonas by phylogenetic analysis of multiple housekeeping genesPseudomonas fluorescensNamed as Pseudomonas fluorescens (Pseudomonas fluorescens) SC 3. And the strain is preserved in Guangdong province microbial strain preservation center in 2018, 4 and 30 days, the preservation number is GDMCC NO.60367, and the preservation address is No. 59 floor 5 floor of the Michelia Tokyo No. 100 college in Guangzhou city.
Example 3 determination of Pseudomonas SC3 bacterial inhibition Spectroscopy
1. In order to better study the biocontrol potential of the strain SC3 of the invention, the bacterial inhibition spectrum of the strain is studied. The specific operation is as follows:
(1) it was investigated whether SC3 has the ability to inhibit the growth of other pathogenic bacteria. Using the same method as that for screening the strain in example 1, the pathogenic bacterium MS2 was replaced with the same amount of rice basal rot EC1, banana bacterial soft rot MS2 and MS3, Ralstonia solanacearum EP1 (15 mL LB in the first layer was replaced with 15 mL TTC), Brassicaceae black rot Xcc, rice bacterial leaf blight Xoo, peach rot pathogenPantoea ananatisPP1, yellow skin rot pathogenPantoea anthophilaCL1, the remaining steps were unchanged.
(2) It was investigated whether SC3 has the ability to inhibit the growth of pathogenic fungi. The fungi to be tested include colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rice sheath blight, rice blast and smut. 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 SC3 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 dried 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 plate, dripping 2 muL of overnight cultured SC3 and Escherichia coli DH5 α bacterial liquid at the same distance from the fungus cakes, sealing after the bacterial liquid is dried, just placing in an incubator at 28 ℃ for culturing for 3-4 days, observing the growth condition of hyphae, and recording the experimental result.
2. Experimental results show that the strain SC3 has strong bacteriostatic action on rice basal rot EC1, banana bacterial soft rot MS2 and MS3, solanaceae ralstonia solanacearum EP1, cruciferae black rot Xcc and rice bacterial blight Xoo, and has strong bacteriostatic action on peach rot pathogenPantoea ananatisPP1, yellow skin rot pathogenPantoea anthophilaCL1 is not ideal in bacteriostasis (as shown in figure 5), has weak bacteriostasis effect on colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rice sheath blight, rice blast and smut (as shown in figure 6), and shows that SC3 can be used for developing biocontrol agents specially aiming at crop bacterial diseases.
Example 4 determination of the Effect of Pseudomonas SC3 on bacterial Soft rot
1. Activating bacterial soft rot MS2 and biocontrol bacteria SC3 strains of banana on an LB solid plate, selecting a single colony in a 50 mL centrifuge tube containing 10 mL LB, placing the centrifuge tube at 28 ℃, shaking the centrifuge tube at 180 rpm for overnight culture, and taking 1 mL OD600About 1.3 of the SC3 bacterial liquid 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, SC3+ LB and SC3+ MS2 mixed liquid by using a 1 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 bacterial strain SC3 can obviously reduce the incidence rate of bacterial soft rot on radish, carrot, Chinese cabbage, potato (shown in figure 7) and banana powder (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 (9)
1. Pseudomonas fluorescens SC3, which is preserved in Guangdong province collection of microorganisms in 2018 at 30 months 4 and with the preservation number of GDMCC NO. 60367.
2. Use of pseudomonas fluorescens SC3 for controlling bacterial diseases in crops, wherein said bacterial diseases in crops comprise bacterial diseases caused by Dickeya zeae or Erwinia.
3. The use according to claim 2, wherein the bacterial disease comprises bacterial soft rot, bacterial basal rot of rice, bacterial wilt of solanaceae, black rot of cruciferae or bacterial leaf blight of rice.
4. Use according to claim 2, wherein the bacterial soft rot disease comprises bacterial soft rot disease on bananas, plantains, juniper, radishes, carrots, chinese cabbage or potatoes.
5. A biological agent for controlling crop bacterial diseases, which comprises pseudomonas fluorescens SC3 of claim 1.
6. The biological agent of claim 5, wherein the fluorescence is measuredPseudomonas sp SC3 at OD6001.0～1.5。
7. A method for controlling bacterial diseases in crops, characterized in that the biological preparation according to claim 6 is inoculated onto plant material.
8. The method of claim 7, wherein the inoculation is by injection inoculation.
9. The method of claim 7, wherein said inoculation is performed by stab inoculation.
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| CN110643538B (en) * | 2019-10-15 | 2021-04-27 | 浙江大学 | Pseudomonas fluorescens XY2F4 and application thereof in preventing and treating crop verticillium wilt |
| 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 |
| CN113293108A (en) * | 2021-05-08 | 2021-08-24 | 四川农业大学 | Preparation method of microbial agent for cleaning orchid pot soil, obtained biological agent and application |
| 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 |
| CN116548473B (en) * | 2023-07-04 | 2023-09-05 | 云南省农业科学院农业环境资源研究所 | Composition containing pseudomonas solanacearum and application of composition in flower diseases |
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