CN117887580B - Protozoan tail trichomonas for promoting banana growth and preventing and controlling fusarium wilt and application thereof - Google Patents
Protozoan tail trichomonas for promoting banana growth and preventing and controlling fusarium wilt and application thereof Download PDFInfo
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Abstract
The invention provides a protozoan tail trichomonas strain for promoting banana growth, preventing and controlling fusarium wilt and application thereof. A protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 for promoting banana growth and preventing and controlling fusarium wilt has a preservation number of CCTCC NO: C202443. Experiments show that the protozoan tail trichomonas NJAU-Z1 disclosed by the invention shows remarkable banana-promoting effect, and meanwhile, the tail trichomonas NJAU-Z1 can directly inhibit the growth of banana fusarium oxysporum, which is a banana fusarium wilt pathogen, and has better control capability on banana fusarium wilt. The invention shows that the protozoan tail trichomonas NJAU-Z1 has great application potential in the aspects of banana growth promotion and banana wilt biological control, and is an important method for realizing agricultural green development and sustainable pest control.
Description
Technical Field
The invention belongs to the technical field of microbiology and plant disease biocontrol, and in particular relates to protozoan tail trichomonas capable of promoting banana growth and preventing and controlling fusarium wilt and application thereof.
Background
Bananas are the major cash crops in world agricultural production and international fruit trade. Banana vascular wilt (Banana WILT DISEASE) caused by Fusarium oxysporum (Fusarium oxysporum f.sp. Cubense, abbreviated as Foc) is a soil-borne vascular bundle disease which seriously endangers Banana and Banana (Musa) production. Foc can survive in the soil and spread by infecting banana roots. After the banana plants are infected by the pathogenic bacteria, the root cells are first necrotized, then the vascular bundles are invaded and blocked, the normal transmission of moisture and nutrients in the plant bodies is blocked, and finally the plants are withered and dead. The early stages of banana wilt appear as scorch at the edges of leaves, and as the disease progresses, the plants gradually lose their normal green color and cause the leaves of the plants to turn yellow and brown in color. Finally, the plants are completely withered and cannot be recovered. Banana vascular wilt severely limits the sustainable development of banana industry.
Aiming at banana wilt, no effective chemical agent is available at present, so that the banana wilt is prevented and controlled mainly in modern agriculture by selecting measures such as disease-resistant varieties, soil improvement, regional isolation, biological prevention and control, rotation and the like. The banana variety for planting banana wilt resistance is an effective means, and aiming at disease-resistant breeding work of banana wilt, the lack of ideal source-resistant materials is slow in progress, and in addition, pathogenic bacteria can develop new organisms to overcome the resistance of the original banana variety, so that the resistant variety needs to be continuously improved. At present, a technical strategy for modifying banana plant genes to enhance the capability of the banana plant genes to resist banana wilt and finally obtaining potential disease-resistant varieties is also available by utilizing a gene editing technology, but the gene editing technology relates to potential risks to an ecological system and human health. The soil structure is improved by adopting measures such as organic matters, biomass covers and the like, so that the biodiversity of a soil ecosystem can be increased, the spread of pathogenic bacteria in soil can be slowed down, but the soil improvement is a long-term process, and the improvement effect is not obvious in a short term due to the requirement of large cost investment. The regional isolation can avoid the banana plants from contacting with the soil infected by pathogenic bacteria, reduce the transmission possibility of the pathogenic bacteria, but under the condition of limited land resources, the establishment of the isolated region can reduce the land utilization efficiency and limit the plant root growth region.
The application of biological control organic fertilizer, biological microbial inoculum and the like can inhibit the survival of pathogenic pathogens by regulating the interaction of antibiotics, competition, re-mailing, lysozyme and the like in the soil microbial population or between the populations so as to achieve the aim of controlling plant diseases. Biological control is mainly aimed at target pathogens, is an eco-friendly agricultural practice, can provide stable pest or disease control over a long period of time, has no soil pollutant residues, and is beneficial to protecting and maintaining the stability of an agricultural ecosystem. In addition, the current research also widely adopts a rotation strategy (such as rotation system of pepper/banana and eggplant/banana rotation) on banana planting, and the banana wilt is prevented and controlled by regulating the number of the soil microorganism core functional microorganisms. On the basis, the application of the Foc biocontrol bacteria in agriculture has extremely high potential for preventing and controlling banana wilt. The biocontrol bacteria can secrete antibacterial substances to inhibit the growth of pathogenic bacteria or inhibit the growth of pathogenic bacteria by means of nutrition competition and the like, and can also produce auxin and the like to promote the growth of plants or induce the immune response of the plants to indirectly alleviate the occurrence of diseases.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a protozoan tail trichomonas strain for promoting banana growth and preventing and controlling banana wilt.
It is another object of the present invention to provide a culture of the protozoan trichomonas.
It is a further object of the present invention to provide the use of the protozoan uromonas.
The aim of the invention can be achieved by the following technical scheme:
The protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 is a tail trichomonas which is separated and screened from rhizosphere soil of test field healthy crop plants naturally developed by a test base of the Nanjing agricultural university, and has extremely strong predatory effect on fusarium oxysporum. According to the characteristics of the cyst and living body of the protozoan tail infusorian NJAU-Z1 and the 18S rDNA gene sequence, the protozoan tail infusorian is identified as Cercomonas directa and is preserved in China center for type culture collection (address: the university of Wuhan, hubei province, with a preservation number of CCTCC NO: C202443) at 1 month 20. The invention researches the control effect and the banana growth promoting capability of the trichomonas on the banana vascular wilt and provides scientific basis for the biological control of the banana vascular wilt and the development and utilization of biocontrol microbial inoculum.
The protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 for promoting banana growth and preventing and controlling fusarium wilt is preserved in China center for type culture collection, the preservation date is 2024, 1 month and 20 days, and the preservation number is CCTCC NO: C202443.
The culture of the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1.
The invention also provides a preparation method of the culture of the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1, the tail trichomonas NJAU-Z1 is inoculated into NMAS liquid culture solution, inactivated escherichia coli (OD=0.04) is added, and the mixture is placed in an incubator at 18-22 ℃ and kept stand for 45-50 hours.
The NMAS culture solution is prepared by the following steps of 0.12 g/L of sodium chloride, 0.0004/g/L of magnesium sulfate heptahydrate, 0.0006/g/L of calcium chloride hexahydrate, 0.142/g/L of sodium phosphate and 0.136/g/L of potassium phosphate.
The application of the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 in promoting banana growth and/or preventing banana wilt.
The application of the culture in promoting banana growth and/or preventing banana wilt.
According to the invention, the protozoan tail trichomonas Cercomonas directa NJAU-Z1 with extremely strong resistance to banana wilt pathogenic bacteria is screened out from rhizosphere separation of healthy crops, and fungus pathogenic bacteria co-culture experiments show that the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 has remarkable control effect on banana wilt, and in addition, the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 can also remarkably promote banana growth. Therefore, the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 has important significance for the restoration of continuous cropping soil and the prevention and treatment of soil-borne diseases.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
FIG. 1 is a photograph of morphological features of the protozoan tail trichomonas NJAU-Z1 dormant capsule (left), reproductive capsule (middle) and vegetative body (right) of the present invention.
FIG. 2 is a phylogenetic tree of the 18S rDNA gene sequence of the trichomonas at NJAU-Z1 of the present invention.
FIG. 3 is a box plot of growth diameter of the colonies of the species Fusarium oxysporum f.sp. Cubense as a function of time for both the end-unit cells NJAU-Z1 and Fusarium oxysporum f.sp. Cubense of the present invention in example 3.
FIG. 4 shows the growth of the colonies of the strain Fusarium oxysporum f.sp. Cubense on four days of the co-cultivation of the PDA plates of example 3 of the present invention with both the Trichomonas NJAU-Z1 and Fusarium oxysporum f.sp. Cubense.
FIG. 5 shows the growth of banana plants inoculated with the treatment group (left in FIG. 5) and the blank group (right in FIG. 5) with Trichomonas tail NJAU-Z1 in example 4 of the present invention.
FIG. 6 is a comparison of fresh weights of bananas in the treated group vaccinated with Trichomonas coccoid NJAU-Z1 and the blank group in example 4 of the present invention.
FIG. 7 is a comparison of banana root length in the treated group inoculated with D.tail NJAU-Z1 and the blank group in example 4 of the present invention.
Biological material preservation information
NJAU-Z1, which is classified and named as protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1, is preserved in China center for type culture Collection, with a preservation address of university of Wuhan, china, a preservation date of 2024, 1 month and 20 days, and a preservation number of CCTCC NO: C202443.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. Three replicates were set up for the experiments in the examples below.
Example 1 protozoan isolation and purification:
The inventor collects rhizosphere soil of healthy plants from banana wilt disease attack field blocks of test bases of Nanjing agricultural university three-layer research institute, the depth range of collection is 0-20cm, and the healthy plants are taken back to a laboratory after being sealed by a sterile self-sealing bag. The target pathogen to be tested is fusarium oxysporum (Fusarium oxysporum f.sp. Cubense) provided by the soil organic fertilizer team at the university of south Beijing agriculture. The fungus medium is PDA (Potato Dextrose Agar) solid medium plates.
The protozoa were isolated by soil dilution and then assayed for antagonism of fusarium oxysporum by liquid co-culture. The specific method comprises the following steps: mixing the rhizosphere soil sample of the healthy crop uniformly, weighing 1g of soil, adding into a centrifuge tube of 50mL and adding sterile deionized water of 30 mL, then placing the centrifuge tube into a shaking table of 250 rpm and 20 ℃ for shaking for 15min, so that the rhizosphere soil is fully mixed uniformly and protozoa in the soil are released. After the centrifuge tube was taken out of the shaker and left to stand for 10min, the supernatant liquid was taken into a 96-well plate and E.coli (OD=0.04) was added as food, followed by cultivation for 2d at 20℃under light-shielding cultivation conditions. Protozoa were grown under inverted microscopes 100×, 200×and400× and subjected to gradient dilution and incubation for 2d at 20 ℃. Finally, single protozoan cells are picked into a new 96-well plate by a capillary tube to obtain pure culture of protozoans.
Example 2 identification of protozoa NJAU-Z1:
The screened protozoa are identified by a method combining morphological observation with molecular biology. PCR reaction System (50. Mu.L System): the primers were 2. Mu.L each, 2 XMix 25. Mu.L, and ddH2O was added to make up to 50. Mu.L. The most commonly used universal primer is P-FLA-F/P-FLA-R. (P-FLA-F: 5'- -CGCGGTAATTCCAGCTCCAATAGC- -3') and (P-FLA-R: 5'- -CAGGTTAAGGTCTCGTTCGTTAAC- -3'). The reaction procedure: pre-denatured at 94℃for 3min, denatured at 94℃for 55s, annealed at 50℃for 50s, extended at 72℃for 1min, extended at 72℃for 10min, and stored at 16℃after 35 cycles. The PCR product was collected by electrophoresis on a 1.5% agarose gel and sent to Sanger sequencing, which was performed by Beijing qing department Biotechnology Co., ltd, and the sequencing result was shown as SEQ ID NO. 1.
In NMAS broth, the protozoan tail-cargo area NJAU-Z1 is active, and the single ended flagella slowly swim (right panel of FIG. 1); the dormant capsule state is round and is in a static state without flagella (left diagram of figure 1); the propagation capsule is circular and in a rotary tremor state without flagella (figure 1).
The obtained 18s rDNA gene sequences were aligned to PR2 (Protist Ribosomal Reference) database and NT (Nucleotide Sequence Database) database of NCBI website using BLAST software, and phylogenetic analysis was performed on isolated protozoa using MEGA software. FIG. 2 is a phylogenetic tree constructed based on the 18S rDNA gene sequence. The tail trichomonas NJAU-Z1 sequencing was the most similar to model protozoan tail trichomonas Cercomonas directa, up to 99.18%, and thus the isolated protozoan NJAU-Z1 was identified as Cercomonas directa.
Example 3 plate co-culture experiments of protozoa NJAU-Z1 inhibiting Fusarium oxysporum banana:
Test fusarium oxysporum banana (Fusarium oxysporum f.sp. Cubense), test protozoa were the protozoan tail trichomonas NJAU-Z1 of example 2, accession number: CCTCC NO: C202443.
Tail trichomonas NJAU-Z1 preparation: the trichomonas coccoides NJAU-Z1 was inoculated into NMAS broth, inactivated escherichia coli (od=0.04) was added, and the mixture was placed in a constant temperature incubator at 20 ℃, and subjected to stationary culture. After 48h, 100. Mu.L of the broth was aspirated and counted under an inverted microscope, and the number of trichomonas tails was diluted to 3X 10 4 CFU/mL. Wherein, the NMAS culture solution has the formula of 0.12 g/L sodium chloride, 0.0004/g/L magnesium sulfate heptahydrate, 0.0006/g/L calcium chloride hexahydrate, 0.142/g/L sodium phosphate and 0.136/g/L potassium phosphate.
Preparation of banana vascular wilt pathogenic bacteria suspension: inoculating preserved banana fusarium oxysporum into a liquid PDB (Potato Dextrose Broth) culture medium, and placing the culture medium in a constant-temperature shaking table for shake culture. After 3d, the culture solution is repeatedly washed for three times by sterile water and filtered by sterilized four layers of gauze to obtain fusarium oxysporum spore liquid, 10 mu L of fusarium oxysporum spore liquid is sucked into a blood cell counting plate and is placed under a positive microscope for counting, and the quantity of fusarium oxysporum spore liquid is diluted to 9 multiplied by 10 4 CFU/mL.
A control group (CK) was set: 10. mu.L of fusarium oxysporum spore solution with a concentration of 9X 10 4 CFU/mL+sterile 10. Mu.L of NMAS medium, and 20. Mu.L of the mixed solution were added dropwise to the PDA solid medium plate for 6 replicates.
Setting a treatment group (L): 10. mu.L of Fusarium oxysporum at a concentration of 9X 10 4 CFU/mL+10. Mu.L of Trichomonas coccyx NJAU-Z1 at a concentration of 3X 10 4 CFU/mL, 20. Mu.L of the mixed liquid was added dropwise to the PDA solid medium plate for 6 replicates.
A total of 12 PDA solid medium plates from the above two experimental groups were placed in a fungus incubator at 28 ℃, and fungus colony growth was observed and recorded daily.
FIG. 3 is a graph of dynamic changes in colony diameter over time for both uromonas NJAU-Z1 and Fusarium oxysporum f.sp. Cubense plates, control and treatment Fusarium oxysporum f.sp. Cubense. FIG. 4 shows colony growth of both Trichomonas NJAU-Z1 and Fusarium oxysporum f.sp. Cubense in plates, fusarium oxysporum f.sp. Cubense on the first day (24 h), second day (48 h), third day (72 h), and fourth day (96 h).
From the above, protozoa NJAU-Z1 can inhibit the growth of Fusarium oxysporum, and trichomonas cauda Cercomonas directa NJAU-Z1 shows an inhibitory effect on banana vascular wilt pathogen Fusarium oxysporum f.sp. Cubense.
Example 4 potted test of trichomonas tail (Cercomonas directa) NJAU-Z1 to promote banana growth:
the test set up two treatments: (1) a blank CK; (2) The treated group of tail infusorians Cercomonas directa NJAU-Z1 was inoculated. Each treatment was repeated 5 times. Neither test treatment was inoculated with pathogenic bacteria.
The test banana was a dwarf banana seedling and the test protozoa was the trichomonas coccoid NJAU-Z1 of example 2, accession number: CCTCC NO: C202443.
Tail trichomonas NJAU-Z1 preparation: the trichomonas coccoides NJAU-Z1 was inoculated into NMAS broth, inactivated escherichia coli (od=0.04) was added, and the mixture was placed in a constant temperature incubator at 20 ℃, and subjected to stationary culture. After 48h, 100. Mu.L of the broth was aspirated and counted under an inverted microscope.
Cultivating banana plants: the dwarf banana seedlings to be tested are derived from Guangxi farm, healthy banana seedlings (uninfected pathogenic bacteria) with the height of 15cm are selected and transplanted into 2-gallon flowerpots, potting soil is derived from a farm in Jiang Ning area of Nanjing city of Jiangsu province, the soil is air-dried and then screened, plant residues are removed, and the soil is used for potting experiments, and does not contain and is not inoculated with pathogenic bacteria. After transplanting, inoculating protozoa cultured by NMAS culture solution into the soil of the treatment group flowerpot, wherein the concentration is 10 3 per gram of soil; the blank group was inoculated with an equal amount of NMAS broth (without protozoa). After one month of greenhouse culture, the growth condition of banana pot culture is observed.
FIG. 5 shows the growth of banana plants inoculated with the tail trichomonas (Cercomonas directa) NJAU-Z1 treatment group (left in FIG. 5) and the blank control group (right in FIG. 5). FIG. 6 shows that in example 4 of the present invention, inoculation of D.tail NJAU-Z1 significantly increases the fresh weight of banana. Figure 7 shows that in example 4 of the present invention, inoculation of uromonas NJAU-Z1 significantly elongated the root length of bananas. The protozoan tail trichomonas NJAU-Z1 can promote the growth of banana seedlings. Therefore, the trichomonas caudalis NJAU-Z1 can obviously promote the growth of banana seedling root systems.
Claims (3)
1. The protozoan tail trichomonas NJAU-Z1 Cercomonas directaNJAU-Z1 for promoting banana growth and preventing and controlling fusarium wilt is preserved in China center for type culture collection, the preservation date is 2024, 1 month and 20 days, and the preservation number is CCTCC NO: C202443.
2. The preparation method of the culture of the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 is characterized by comprising the steps of inoculating the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 into NMAS culture solution, adding inactivated microorganisms as food, placing in an incubator at 18-22 ℃, and standing for 45-50h; the protozoan tail trichomonas NJAU-Z1 Cercomonas directa NJAU-Z1 is preserved in China center for type culture collection, the preservation date is 2024, 1 month and 20 days, the preservation number is CCTCC NO: C202443, the NMAS culture fluid is prepared by the preparation method of sodium chloride 0.12 g/L, magnesium sulfate heptahydrate 0.0004 g/L, calcium chloride hexahydrate 0.0006 g/L, sodium phosphate 0.142 g/L and potassium phosphate 0.136 g/L, and the inactivated microorganism is inactivated escherichia coli.
3. Use of the protozoan tail gas of claim 1, NJAU-Z1 Cercomonas directa NJAU-Z1 for promoting banana growth and/or controlling banana vascular wilt.
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| CN116948829A (en) * | 2023-05-31 | 2023-10-27 | 南京农业大学 | Protozoon tail trichomonas NJAU-ZG8 for promoting tomato growth and preventing and controlling tomato bacterial wilt and application thereof |
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| RU2180169C2 (en) * | 1999-08-16 | 2002-03-10 | Черников Андрей Михайлович | Method to protect plants against bacterial and fungous diseases |
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| CN113943660A (en) * | 2021-10-08 | 2022-01-18 | 南京农业大学 | Talaromyces fungus NJAU-L8 for preventing and controlling continuous cropping soil-borne blight and application thereof |
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