CN117887580A - 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. Protozoan tail trichomonas NJAU-Z1 for promoting banana growth and preventing and controlling fusarium wiltCercomonas directaNJAU-Z1 has a preservation number of CCTCC NO: C202443. Experiments show that the protozoan tail trichomonas NJAU-Z1 provided 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 pathogenic bacterium of banana fusarium wilt, and has better control capability on banana fusarium wilt. The invention shows that the tail drop of protozoaThe insect NJAU-Z1 has great application potential in the aspects of banana growth promotion and banana vascular 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. Is prepared from fusarium oxysporumFusarium oxysporum f.sp. CubenseBanana vascular wilt (Banana Wilt Disease) caused by Foc for short) is a soil-borne vascular bundle disease which seriously damages 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 biocontrol bacteria aiming at Foc in agriculture has extremely high prevention and control potential of banana vascular 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 invention relates to protozoan tail trichomonas NJAU-Z1Cercomonas directaNJAU-Z1 is a trichomonas strain which is separated and screened from rhizosphere soil of healthy crop plants of a test field block for natural morbidity of a test base of Nanjing agricultural university, and has extremely strong predatory effect on fusarium oxysporum. Based on the characteristics of the cyst and living body of the protozoan tail trichomonas NJAU-Z1 and the gene sequence analysis of 18S rDNA, the method is identified asCercomonas directaAnd is preserved in 2024 at 1 month and 20 days to China center for type culture Collection (address: university of Wuhan collection in Wuhan district, wuhan, hubei province, with a preservation number of CCTCC NO: C202443). 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.
Protozoan tail trichomonas NJAU-Z1 for promoting banana growth and preventing and controlling fusarium wiltCercomonas directaNJAU-Z1 is preserved in China center for type culture Collection with a preservation date of 2024, 1 month and 20 days, and a preservation number of CCTCC NO: C202443.
The invention relates to a protozoan tail trichomonas NJAU-Z1Cercomonas directaCultures of NJAU-Z1.
The invention also provides the protozoan tail trichomonas NJAU-Z1Cercomonas directaThe preparation method of the culture of the NJAU-Z1 comprises inoculating the tail trichomonas NJAU-Z1 into NMAS liquid culture solution, adding inactivated escherichia coli (OD=0.04), placing into an incubator at 18-22 ℃, and standing for 45-50h.
The NMAS culture solution is prepared with 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.
Said protozoaTail trichomonas NJAU-Z1Cercomonas directaUse of NJAU-Z1 for promoting banana growth and/or for controlling banana vascular wilt.
The application of the culture in promoting banana growth and/or preventing banana wilt.
The invention screens out protozoan tail trichomonas with extremely strong resistance to banana fusarium wilt pathogenic bacteria from healthy crop rhizosphere separationCercomonas directa The NJAU-Z1 fungus pathogen co-culture test shows that the tail trichomonas protozoans NJAU-Z1Cercomonas directaNJAU-Z1 has remarkable control effect on banana vascular wilt, and in addition, the protozoan tail trichomonas NJAU-Z1Cercomonas directaNJAU-Z1 also significantly promotes banana growth. Thus, the protozoan tail trichomonas NJAU-Z1 of the inventionCercomonas directaThe NJAU-Z1 has important significance for restoring continuous cropping soil and preventing and treating 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 showing morphological characteristics 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 sequences of the trichomonas NJAU-Z1 of the present invention.
FIG. 3 shows the tail trichomonas NJAU-Z1 and the tail trichomonas strain in example 3 of the present inventionFusarium oxysporum f.sp. CubenseThe two PDA plates are co-cultured,Fusarium oxysporum f.sp. Cubensebox plot of colony growth diameter versus time.
FIG. 4 shows the tail trichomonas NJAU-Z1 and the tail trichomonas in example 3 of the present inventionFusarium oxysporum f.sp. CubenseThe two PDA plates are co-cultured,Fusarium oxysporum f.sp. Cubensegrowth of colonies within four days.
FIG. 5 shows the growth of banana plants inoculated with the tail trichomonas NJAU-Z1 treatment group (left in FIG. 5) and the blank group (right in FIG. 5) in example 4 of the present invention.
FIG. 6 is a comparison of fresh weights of bananas in the treated group vaccinated with Trichomonas 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 vaccinated with Trichomonas NJAU-Z1 and the blank in example 4 of the present invention.
Biological material preservation information
NJAU-Z1, classified and named as protozoan tail trichomonas NJAU-Z1Cercomonas 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 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 oxysporumFusarium oxysporum f.sp. Cubense) Is provided by the soil organic fertilizer team of Nanjing agricultural university. The fungus medium was a PDA (Potato Dextrose Agar) solid medium plate.
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: the healthy crop rhizosphere soil sample is evenly mixed, 1g of soil is weighed and added into a centrifuge tube of 50 mL, sterile deionized water of 30 mL is added, then the centrifuge tube is placed into a shaking table of 250 rpm and 20 ℃ to oscillate for 15min, so that the rhizosphere soil is fully evenly mixed 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 culture solution, protozoan tail trichomonas NJAU-Z1 presents active state, and single ended flagella slowly swim (right diagram of figure 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. Tail trichomonas NJAU-Z1 sequencing results and model protozoan tail trichomonasCercomonas directaThe similarity was highest, up to 99.18%, and thus the isolated protozoa NJAU-Z1 was identified asCercomonas directa。
Example 3 plate co-culture experiments of protozoa NJAU-Z1 inhibiting Fusarium banana:
sample fusarium oxysporum f.bananaFusarium oxysporum f.sp. Cubense) The protozoa tested were the protozoa tail trichomonas NJAU-Z1 of example 2, accession number: CCTCC NO: C202443.
Tail trichomonas NJAU-Z1 preparation: the tail trichomonas NJAU-Z1 was inoculated into NMAS culture medium, 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 culture 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 a sterilized four-layer 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 normal 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 concentration is 9X 10 4 CFU/mL fusarium oxysporum spore solution + sterile 10. Mu.L NMAS broth, 20. Mu.L of the mixed solution was added dropwise to the PDA solid medium plate for 6 replicates.
Setting a treatment group (L): 10. mu.L concentration is 9X 10 4 CFU/mL Fusarium oxysporum+10. Mu.L at a concentration of 3X 10 4 CFU/mL of uromonas NJAU-Z1, 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 diagram of the tail trichomonas NJAU-Z1 andFusarium oxysporum f.sp. Cubenseco-culture of both plates, control and treatmentFusarium oxysporum f.sp. CubenseDynamic plot of colony diameter over time. FIG. 4 is a diagram of a tail trichomonasNJAU-Z1Fusarium oxysporum f.sp. CubenseThe two plates are co-cultured,Fusarium oxysporum f.sp. Cubensecolony growth on the first day (24 h), second day (48 h), third day (72 h), and fourth day (96 h).
From the above, the protozoa NJAU-Z1 can inhibit the growth of Fusarium oxysporum, and the tail trichomonasCercomonas directa NJAU-Z1 develops a pathogen against banana vascular wiltFusarium oxysporum f.sp. CubenseIs a compound having an inhibitory effect on the inhibition of the activity of the polypeptide.
EXAMPLE 4 Tail trichomonasCercomonas directa）Potting test for banana growth promoted by NJAU-Z1:
the test set up two treatments: (1) a blank CK; (2) Inoculating tail trichomonasCercomonas directa Treatment group of NJAU-Z1. 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 tail trichomonas NJAU-Z1 of example 2, accession number: CCTCC NO: C202443.
Tail trichomonas NJAU-Z1 preparation: the tail trichomonas NJAU-Z1 was inoculated into NMAS culture medium, 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, the treated flower pot soil is inoculated with protozoa cultured by NMAS culture solution with the concentration of 10 3 Each 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 inoculation of tail trichomonasCercomonas directa）Growth status of banana plants in the treated group of NJAU-Z1 (left in FIG. 5) and in the blank group (right in FIG. 5). FIG. 6 shows that in example 4 of the present invention, the inoculation of the tail trichomonas NJAU-Z1 significantly increases the fresh weight of the banana. Figure 7 shows that in example 4 of the present invention, the root length of banana was significantly elongated by inoculating the tail trichomonas NJAU-Z1. 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 (5)
1. Protozoan tail trichomonas NJAU-Z1 for promoting banana growth and preventing and controlling fusarium wiltCercomonas directaNJAU-Z1 is preserved in China center for type culture Collection with a preservation date of 2024, 1 month and 20 days, and a preservation number of CCTCC NO: C202443.
2. Protozoan tail trichomonas NJAU-Z1Cercomonas directaA process for producing a culture of NJAU-Z1, characterized by comprising culturing the protozoan uromonas NJAU-Z1Cercomonas directaInoculating NJAU-Z1 into NMAS culture solution, adding inactivated microorganism as food, placing into an incubator at 18-22deg.C, and standing for 45-50 hr; the protozoan tail trichomonas NJAU-Z1Cercomonas directaNJAU-Z1 is preserved in China center for type culture Collection with a preservation date of 2024, 1 month and 20 days, and a preservation number of CCTCC NO: C202443.
3. The preparation method according to claim 2, wherein the NMAS culture solution is prepared by a 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.
4. The method of claim 2, wherein the inactivated microorganism is inactivated escherichia coli.
5. The protozoan tail of claim 1, NJAU-Z1Cercomonas directaNJAU-Z1 is used for promoting banana growth andand/or preventing and treating banana vascular wilt.
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