CN116496941B - Microbial fermentation compound and application thereof in preventing and treating citrus yellow dragon disease - Google Patents
Microbial fermentation compound and application thereof in preventing and treating citrus yellow dragon disease Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/07—Bacillus
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
The application discloses a microbial fermentation compound and application thereof in preventing and controlling citrus yellow dragon disease, and relates to the technical field of plant disease prevention and control. The microbial fermentation compound is obtained by adding microorganisms into a mixture of pig manure, bran and soybean meal powder for fermentation. The application also discloses a method for treating citrus seedlings by adopting the microbial fermentation compound, and the method can effectively prevent and treat citrus yellow dragon disease; the yellowing and mottle symptoms of the citrus seedlings treated by the method are reduced or the mottle tends to disappear; the citrus seedling leaves have smooth surfaces and strong appearance; the citrus seedlings have the advantages of large number of new roots, large quantity of branches, luxuriant growth of branch tips and obvious rejuvenation effect.
Description
Technical Field
The application relates to the technical field of plant disease prevention and control, in particular to a microbial fermentation compound and application thereof in preventing and controlling citrus yellow dragon disease.
Background
Citrus yellow dragon disease is a destructive disease in world citrus production, caused by a gram-negative bacterium that is restricted to the phloem-parasitic, and is capable of infecting a variety of rutaceae plants including citrus, trifoliate, kumquat, murraya, and the like. Currently, the disease is mainly distributed in the near 50 countries and regions of asia, africa, oceangoing, south america and north america, severely restricting the healthy development of the citrus industry.
As the yellow dragon disease pathogen can not be cultured in vitro, the research on the pathogenic mechanism of the phloem bacillus and the disease resistance of the citrus is difficult, and the prevention and the treatment of the yellow dragon disease still become a worldwide difficult problem. The yellow dragon disease has long incubation period and complex symptoms, even the concentration of pathogenic bacteria changes in different tissues, different parts and different seasons of a tree, no effective prevention and control means are found for the yellow dragon disease of citrus, and the yellow dragon disease is difficult to thoroughly clear, and can be prevented and controlled only by digging out the disease tree, killing psyllids and cultivating disease-free seedlings. When the chemical prevention and control of psyllids are carried out, environmental pollution is easy to cause, drug resistance is generated, and the concept of sustainable development is not met.
The biological control can make up the defects of chemical control, does not pollute the environment, does not destroy ecological balance, does not generate drug resistance, is also efficient, low in cost and convenient to use, and has the advantage of sustainable development. The effective biological control means can be constructed to effectively solve the problem that the citrus yellow dragon disease is difficult to treat.
Disclosure of Invention
Biological control of plant diseases refers to control of plant diseases using beneficial microorganisms (biocontrol bacteria) including mainly bacteria, fungi, actinomycetes and viruses or metabolites of the microorganisms; the main biological control mechanisms of biocontrol bacteria include: antagonism, competition, re-mailing, inducing plant resistance, promoting plant growth, etc. The biocontrol bacteria can be under the combined action of multiple mechanisms under the field condition, and can also act on different parts of plants in different development periods, so as to achieve the effects of inhibiting the growth and the reproduction of pathogenic bacteria or killing the pathogenic bacteria. Antagonism refers to the inhibition, even killing, of the activity of a secondary metabolite produced by a microorganism during its life activity on other microorganisms; the competition effect means that two or more microorganisms compete for living space, sites, nutrition, moisture and the like, and the biocontrol bacteria can become dominant bacteria in plants so as to limit the growth of other pathogenic bacteria; heavy parasitism refers to the phenomenon that pathogenic microorganisms parasitic on plants are again parasitized by other microorganisms. Plant resistance is generated by two induction routes, namely systemic acquired resistance (Systemic acquired resistance, SAR), namely resistance to pathogens is induced by unvaccinated parts after pathogen infection; second, induced resistance (Induced systemic resistance, ISR), refers to the enhancement of resistance by the physical or chemical barrier that the plant body produces after stimulation of the plant by non-pathogenic agents.
The method utilizes the microbial fermentation complex 'SS' to continuously treat citrus seedlings for a plurality of times, adopts a recognized high-sensitivity real-time quantitative QPCR detection method to track and detect the relative expression quantity of the RNR gene of the phloem bacillus, and simultaneously compares and observes the phenotype characteristics of the seedlings, so as to confirm the effects of the microbial fermentation complex 'SS' in killing the phloem bacillus and promoting the rejuvenation of the seedlings, and provides a practical method for gradually clearing the citrus yellow dragon disease pathogen in the seedlings and obtaining healthy and disease-free citrus female parent seedlings. For this reason, the application provides following technical scheme:
(1) A microbial fermentation complex "SS" obtained by the following method:
uniformly mixing pig manure, bran and bean pulp powder according to a mass ratio of 90:5:5 to obtain a first mixture; adding microorganisms and water into the first mixture, and mixing to obtain a second mixture, wherein the water content of the second mixture is controlled between 60 and 65 percent;
piling and fermenting at 25-34 ℃, turning over for 1 time every 2 days, and continuously fermenting for 21 days; i.e.the microbial fermentation complex "SS" is obtained.
The microbial fermentation complex "SS" of (1), wherein the microorganism comprises at least one of the microorganisms of (a) and (b): (a) Bacillus sp TD 3-1; (b) Bacillus sp TD 3-2.
Among the microorganisms mentioned above, the two microorganisms, "Bacillus sp TD 3-1" and "Bacillus sp TD 3-2" are both preserved, the preservation unit is China center for typical culture Collection, the address is located in eight-channel 299 of Wuchang district of Wuhan, hubei province, the preservation numbers are respectively CCTCC NO: M2020563 and CCTCC NO: M2020564, and the preservation dates are 2020, 9 months and 30 days.
(2) A method of controlling citrus yellow dragon disease comprising:
obtaining a microbial fermentation complex "SS" as described in (1);
1 time every 7 days, and 5-20 g/plant of the plant is applied to the seedling pot each time;
loosening the pot soil 1 time every 2 weeks;
1 granular compound fertilizer is applied every 2 weeks, wherein the compound fertilizer N is P, K=15:15:15, and the application amount is 5 g/plant;
1 time of chelation type medium trace elements are applied every 4 weeks, wherein the trace elements contain silicon, calcium, boron, zinc, iron and potassium and are applied to the basin according to the amount of 1 gram/plant;
daily management: applying seaweed organic fertilizer for 2 times; the potted seedlings are watered conventionally, and the soil is kept moist.
(3) The microbial fermentation complex SS and the application of the method in preventing and treating citrus yellow dragon disease.
The technical effects of the microbial fermentation compound and the application thereof in preventing and treating citrus yellow dragon disease are specifically illustrated in the examples.
Drawings
FIG. 1 shows the variation of shoot characteristics of citrus seedlings by the microbial fermentation complex "SS" provided in the examples of the present application, wherein the total of three citrus seedlings before and after the treatment; wherein A, D is the first and pre-treated citrus seedlings, B, E is the second and pre-treated citrus seedlings, and C, F is the third and pre-treated citrus seedlings.
FIG. 2 is a comparison of microbial fermentation complex "SS" treatment provided in the examples herein with control nursery stock growth; wherein A is the nursery stock of the treatment group, and B is the nursery stock of the control group.
FIG. 3 is a graphical representation of the leaves of 3-4 year old citrus seedlings treated with microbial fermentation complex "SS" in excess as provided in the examples herein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
In the embodiment of the application, bacillus sp TD 3-1 and Bacillus sp TD 3-2 are two Bacillus strains, the Bacillus sp TD 3-2 is screened from a deposit around a pig fine breed breeding base of a university of China agricultural university, the two strains are preserved, the preservation unit is China center for typical culture, the address is in eight path 299 No. of Wuchang area of Wuhan, hubei province, the Bacillus sp TD 3-1 and the preservation number is CCTCC NO: M2020563, the preservation number of the Bacillus sp TD 3-2 is CCTCC NO: M2020564, and the preservation date is 2020, 9 months and 30 days.
In the embodiment of the application, the bacillus strain is derived from a heap around a pig fine breed breeding base of the university of China agricultural university.
Microbial fermentation complex "SS"
In the embodiment of the application, the microbial fermentation compound "SS" is obtained by adding a microbial fermentation to a first mixture, wherein the first mixture is formed by mixing pig manure, bran and soybean meal. In some embodiments, the mass ratio of the pig manure, the bran and the soybean meal is 90:5:5.
In embodiments of the present application, the microorganism may comprise one or two, in particular, at least one of (a) Bacillus sp TD 3-1 (CCTCC NO: M2020563) and (b) Bacillus sp TD 3-2 (CCTCC NO: M2020564). In some preferred embodiments, the microorganism comprises (a) and (b) 2.
In the embodiment of the application, the microorganism, the first mixture and the water form a second mixture, wherein the mixing mass ratio of the microorganism to the first mixture is 8:1000, such as 10kg of the first mixture is added to 80g of the microorganism for mixing; in the second mixture, the water content is controlled to be 60-65%.
In the embodiment of the application, the second mixture is subjected to heap fermentation at 25-34 ℃ for a fermentation period of 21 days. In some preferred embodiments, the second mixture is flipped 1 time every 2 days during the fermentation cycle to achieve better fermentation.
Method for preventing and treating citrus yellow dragon disease
In the embodiment of the application, a method for preventing and treating citrus yellow dragon disease is provided, namely, citrus seedlings are continuously treated for a plurality of times through the microbial fermentation complex 'SS'. In some embodiments, citrus seedlings used are selected from 3-year-old Nanfeng citrus seedlings purchased from Jiangxi (stock is fructus Aurantii), and 7-gallon basin-planted seedlings are used. The seedlings are placed in the China agricultural university agricultural microorganism national engineering center net room for isolated planting. The culture medium is as follows: 50% of vegetable garden soil, 25% of peat and vermiculite respectively.
In some embodiments, the microbial fermentation complex "SS" treats citrus seedlings by: firstly, 1 time every 7 days, each time in an amount of 5-20 g/plant, in one embodiment, 1 time every 7 days of microbial fermentation complex "SS" is applied for 25 times during the period from 7 months in 2023 to 1 month in 2023; in a preferred embodiment, the microbial complex "SS" is applied to the pot in an amount of 5 g/plant. Next, 1 pot of soil was loosened every 2 weeks, and 1 granular compound fertilizer was applied every 2 weeks, wherein compound fertilizer N: P: k=15:15:15 (N-P 2 O-K 2 O total oxygen fraction>45%) and the application rate was 5 g/strain.
In some embodiments, the process of treating citrus seedlings with microbial fermentation complex "SS" further comprises: the chelating medium trace elements containing Si, ca, B, zn, fe and K were applied 1 time every 4 weeks to the pot in an amount of 1 g/plant.
In the embodiment of the application, the treatment process of the microbial fermentation compound 'SS' on the citrus seedlings also comprises daily management, namely, application of the seaweed organic fertilizer for 2 times during the treatment; the potted seedlings are watered conventionally, and the soil is kept moist.
Microbial fermentation complex "SS" and application of treatment method thereof
The embodiment of the application also provides the microbial fermentation complex SS and application of the microbial fermentation complex SS to a processing method of citrus seedlings, verification analysis is carried out on the processing result, the verification analysis is realized by adopting a method for carrying out QPCR detection on the phloem bacillus of citrus yellow dragon disease, and molecular detection of pathogen is mainly carried out by collecting veins and branch and bark of citrus. In one embodiment, the sampling time before treatment is 2022, 7 months and 15 days, the sampling time after treatment is 2022, 12 months and 6-9 days, and the sample is stored at-80 ℃ and is used for detection.
In certain embodiments of the present application, total DNA from a citrus sample is extracted using the CTAB method and the DNA mass is detected using a nucleic acid protein meter. In the test process, the titer change of the leaf vein detection yellow dragon disease pathogen is collected, so that the verification result of the microbial fermentation complex SS after the citrus processing is obtained.
In the implementation of the application, the reaction system and the reaction procedure of the QPCR detection method are shown in the following table 1;
TABLE 1
| Reaction system | System (mul) | Reaction procedure |
| 2×SYBR PCR mix | 5 | 94℃，5min |
| DNA | 0.5 | 94℃，15s |
| Forward primer(10μm) | 0.1 | 60℃，20s |
| Revese primer(10μm) | 0.1 | 72℃，20s |
| ddH 2 O | 4.3 | 2to 4for 40cycles |
| 72℃，10min | ||
| 40℃，10s |
The main instruments used in the test are shown in table 2 below.
TABLE 2
| Main instrument | Belonging to the company |
| Fluorescent quantitative PCR instrument | Corbett RG 6000 |
| Conventional PCR instrument | Bio-Rad Co Ltd |
| Nucleic acid protein tester | Amersham Co |
| DYY-5 type electrophoresis apparatus | Beijing Liuyi Instrument Factory |
| Gel imaging instrument | Segate Co Ltd |
| High pressure sterilizing pot | German System Co |
| Super clean bench | SHANGHAI MICRO-X FURNACE INDUSTRY Co.,Ltd. |
| Digital display constant temperature water bath HH-Z | National Electrical Co Ltd |
| Illumination incubator | Ningbo sea-block blessing laboratory instrument |
| GNP-9080 type constant temperature incubator | Shanghai precision macro laboratory Equipment Co.Ltd |
| Electronic balance | Shanghai flower tide electric appliances Co Ltd |
| Full-temperature oscillator HZQ-Q | Harbin, east Linn electronic Co.Ltd |
| System biological microscope | NIKON instruments (Shanghai Co., ltd.) |
| Fast-prep tissue grinder | MP biomedicals Co |
| Multifunctional enzyme labeling instrument | Perkinelmer Co |
In the embodiment of the application, a PCR detection template takes DNA of an extracted vein sample as a template, a citrus plant cytochrome oxidase gene COX (Li et al, 2006) is an internal reference gene, and a primer is COX+/COX-; the target gene was amplified using the high sensitivity yellow dragon disease primer RNR+/RNR- (Table 3), which is a very sensitive recognized primer in yellow dragon disease detection, with 5 copies. Each sample was set up in 3 replicates. Healthy "newhol" callus DNA was used as negative control, 2 -△△CT The value was set to 1 (Li et al, 2006). The "sugar orange" leaf DNA detected as positive plants by the laboratory was used as a positive control for QPCR. Test data using 2 -△△CT Analysis by the method, if sample 2 -△△CT A value greater than 1 is considered a positive plant. Data were analyzed by anova and significance level using Excel 2016 software and SPSS 25.0 software.
TABLE 3 Table 3
| Primer(s) | Nucleic acid sequence (5 '-3') | Genebank numbering |
| COX+ | The sequence is shown as SEQ ID NO.1 | CX297817 72-93 |
| COX- | The sequence is shown as SEQ ID NO.2 | CX297817 118-139 |
| RNRf | The sequence is shown as SEQ ID NO.3 | CP010804.1 5434-5455 |
| RNRr | The sequence is shown as SEQ ID NO.4 | CP010804.1 5493-5514 |
In the embodiment of the application, the adopted fluorescence quantitative detection method is relative quantitative detection, specifically adopts a "-delta CT method", wherein:
CT value: cycle Threshold, number of amplification cycles. Representing the number of amplification cycles undergone by the fluorescent signal in each PCR reaction tube to reach a set threshold;
Δct (gene of interest) =ct (gene of interest of test cells) -CT (gene of interest of control cells);
Δct (reference gene) =ct (test cell reference gene) -CT (control cell reference gene);
ΔΔΔCT =Δct (Gene of interest) - Δct (reference gene);
ratio of expression level of target gene in test cell to control cell=2 -ΔΔCT ；
Negative control 2 -△△CT When the CT value of the sample detection result is calculated, the relative expression amount is 2 -△△CT >1, the plants are positive and have yellow dragon disease pathogen; when 2 -△△CT <1, the sample is pathogen free.
The present application also provides a control example, i.e., the microbial fermentation complex "SS" is applied to citrus seedlings at an amount exceeding 20 g/plant, 21 g/plant, at elevated temperature (7 months of 2022), and other treatments and detection methods are the same as in the previous examples.
Results:
(1) Phenotypic changes in citrus seedlings after microbial fermentation complex "SS" treatment.
After citrus seedlings were treated with the microbial fermentation complex "SS" provided by the examples of the present application, the phenotype of the citrus leaves was significantly changed. Observations find that: positive seedlings from the treatment group exhibited typical yellowing mottle symptoms prior to treatment (as shown in fig. 1A), and from the beginning of the treatment with microbial fermentation complex "SS" in the 7 th of 2022, the yellowing mottle symptoms on the leaves were reduced or the mottle tended to disappear when treated 10 times per week (as shown in fig. 1B), and the leaves turned green, were smooth in surface, waxy and robust in appearance when continued to 20 times of treatment (as shown in fig. 1C). The leaves of the yellow dragon positive control plants can be observed to have different degrees of yellowing mottle symptoms on the tips of the branches, and the leaves become mature along with the lengthening of the growth period, but the top leaves still show the characteristic of yellowing mottle (shown in figures 1D-F). This demonstrates that microbial fermentation complex "SS" plays a significant role in turning green leaves of seedlings.
From the growth condition of the whole plant of the nursery stock, the nursery stock after being treated by the microbial fermentation compound SS has developed root systems, more new roots, more branches, luxuriant growth of branch tips (as shown in the method of figure 2A), and the nursery stock root system of the control group has browning phenomenon, and the nursery stock without treating the plant has strong growth condition (as shown in figure 2B). Thus, the microbial fermentation complex SS has good effect on citrus seedling rejuvenation.
(2) Effects of microbial fermentation complex "SS" treatment on the bacillus phloem titer.
In the embodiment of the application, QPCR is adopted to detect the key pathogenic gene RNR design primer of the phloem bacillus pathogen, and 6 biological repeats are respectively arranged in the treatment group and the control group. The results show that: when the seedlings are treated by the microbial fermentation compound 'SS', the positive rate of the veins of the treated group samples is changed from 5/6 to 1/6, the positive rate of the branches and barks is changed from 4/6 to 2/6, and as a result of comprehensively evaluating the titer of the yellow dragon disease, the positive rate before treatment accounts for 5/6 (83.3%), the positive rate after treatment is 2/6 (33.3%) after 20 times, and the positive rate is reduced by 50%. The positive rate of the veins in the control group at the beginning of the test was 1/6, the positive rate of the branch skin in the control group was 3/6, the positive rate at the beginning of the comprehensive evaluation test was 4/6 (66.6%), the positive rate of the veins in the control group was still 1/6 at the time point of 20 treatments, the positive rate of the branch skin was changed from 3/6 (66.6%) to 5/6 (83.3%), the comprehensive evaluation positive rate was 5/6 (83.3%), and the detailed results are shown in Table 4 below.
TABLE 4 Table 4
(3) The pathogenic phloem bacillus of the yellow dragon disease exists mainly in phloem. The concentration of pathogenic bacteria fluctuates due to different tissues and seasons of each tree. In order to accurately judge the virulence condition of the phloem bacillus pathogen in the nursery stock, the embodiment of the application carries out tracking detection on the veins and the branch barks. From the change trend of the yellow dragon disease pathogen, the treatment of the microbial fermentation complex SS can convert 50% of positive materials into negative materials, and is effective for reducing the pathogen titer of the phloem bacillus and removing the pathogen on part of seedlings.
(4) Previous studies have also shown that the phloem bacillus pathogen concentration is at a higher level in infected seedlings during autumn and winter, with the yellowing mottle symptoms of the leaves being most pronounced. In the examples of the present application, the results of sampling and detection at 12 months 6 to 9 days 2022 after the treatment with the microbial fermentation complex "SS" are more indicative that the microbial fermentation complex "SS" can withstand the test in autumn and winter.
(5) In seasons with higher air temperature, the temperature reaches about 38 ℃, and the usage amount of the microbial fermentation compound "SS" for 3-4 years of citrus seedlings is preferably not more than 20 g/plant each time. Excessive use amount can cause partial leaf withering and falling (shown in figure 3), and meanwhile, attention is paid to the matched use of compound fertilizer, medium trace elements and organic fertilizer so as to prevent nutrition competition of various microorganisms and citrus plants.
In summary, the microbial fermentation complex "SS" and the method for treating citrus seedlings with the microbial fermentation complex "SS" provided by the present application can significantly improve the growth state of citrus seedlings. The analysis and evaluation of the phloem bacillus of the citrus Miao Shemai and the branch skin are carried out by a QPCR molecular detection method, and the result shows that the positive rate of the citrus yellow dragon disease can be reduced by 50% after the continuous treatment of the microbial fermentation compound SS treatment for 20 times, the research provides an advantageous female parent material for the cultivation of the citrus disease-free seedlings, and a practical, safe and effective means for controlling and eliminating the phloem bacillus is provided, so that the method has great application value and wide market prospect for rejuvenation of citrus trees.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (3)
1. An application of microorganism fermentation complex in preventing and treating citrus yellow dragon disease;
the preparation method of the microbial fermentation complex comprises the following steps:
obtaining a first mixture, wherein the first mixture comprises pig manure, bran and soybean meal powder in a weight ratio of 90:5:5;
adding microorganisms and water into the first mixture for mixing to obtain a second mixture, wherein the mixing mass ratio of the first mixture to the microorganisms is 1000:8, and the water content of the second mixture is controlled between 60 and 65 percent; and
piling up and fermenting the second mixture at 25-34 ℃ and turning over for 1 time every 2 days, and continuously fermenting for 21 days; obtaining the microbial fermentation complex;
wherein the microorganism isBacillus sp. TD 3-1Bacillus spTD 3-2; the saidBacillus sp. TD 3-1 is preserved in China center for type culture Collection (China university of Wuhan, china) at 9 months and 30 days in 2020, and has a preservation number of CCTCC NO: M2020563; the saidBacillus spTD 3-2 is preserved in China center for type culture Collection (China university of Wuhan, china) at 9 months and 30 days in 2020, and has a preservation number of CCTCC NO: M2020564;
the steps of the application include:
and applying the microbial fermentation compound to citrus seedling pots according to the amount of 5-20 g/plant every 7 days for 1 time, and loosening pot soil every 2 weeks.
2. The use according to claim 1, further comprising applying 1 granular compound fertilizer every 2 weeks, wherein N: P: k=15:15:15 in the compound fertilizer is applied at a rate of 5 g/plant.
3. The use according to claim 1, said steps further comprising daily management, i.e. applying 2 seaweed organic fertilizer during the period; the potted seedlings are watered conventionally, and the soil is kept moist.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483053A (en) * | 2016-01-04 | 2016-04-13 | 江西天人生物控股有限公司 | Microbial compound bacteria for preventing and treating citrusyellowshoot and preparation method and application thereof |
| WO2018014169A1 (en) * | 2016-07-19 | 2018-01-25 | 张铭强 | Method for preventing and controlling citrus greening disease and yellows disease by applying biological carbon fertilizer |
| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
| CN109306334A (en) * | 2018-10-29 | 2019-02-05 | 云南省微生物发酵工程研究中心有限公司 | One plant of Bei Laisi bacillus L1-21 and its prevention and treatment Citrus Huanglongbing pathogen application |
| CN111837766A (en) * | 2020-07-27 | 2020-10-30 | 华中农业大学 | Method for controlling pathogen of seedling phlobacterium phlogistii by composite microorganism treatment and application |
| CN113667620A (en) * | 2021-09-15 | 2021-11-19 | 南宁汉和生物科技股份有限公司 | Bacillus subtilis Bs1218m and its application |
-
2023
- 2023-04-04 CN CN202310352978.3A patent/CN116496941B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483053A (en) * | 2016-01-04 | 2016-04-13 | 江西天人生物控股有限公司 | Microbial compound bacteria for preventing and treating citrusyellowshoot and preparation method and application thereof |
| WO2018014169A1 (en) * | 2016-07-19 | 2018-01-25 | 张铭强 | Method for preventing and controlling citrus greening disease and yellows disease by applying biological carbon fertilizer |
| CN108220185A (en) * | 2017-12-19 | 2018-06-29 | 华中农业大学 | A kind of application of biological control agent in citrus treelet yellow twig cause of disease is removed |
| CN109306334A (en) * | 2018-10-29 | 2019-02-05 | 云南省微生物发酵工程研究中心有限公司 | One plant of Bei Laisi bacillus L1-21 and its prevention and treatment Citrus Huanglongbing pathogen application |
| CN111837766A (en) * | 2020-07-27 | 2020-10-30 | 华中农业大学 | Method for controlling pathogen of seedling phlobacterium phlogistii by composite microorganism treatment and application |
| CN113667620A (en) * | 2021-09-15 | 2021-11-19 | 南宁汉和生物科技股份有限公司 | Bacillus subtilis Bs1218m and its application |
Non-Patent Citations (1)
| Title |
|---|
| 柑橘黄龙病株不同部位内生细菌群落结构的多样性;刘波等;生态学报;第31卷(第24期);第7325-7342页，参见全文 * |
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