CN114214210A - Method for rapidly preparing maize puccinia polycephala bacterial liquid in batches - Google Patents
Method for rapidly preparing maize puccinia polycephala bacterial liquid in batches Download PDFInfo
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- CN114214210A CN114214210A CN202111612546.9A CN202111612546A CN114214210A CN 114214210 A CN114214210 A CN 114214210A CN 202111612546 A CN202111612546 A CN 202111612546A CN 114214210 A CN114214210 A CN 114214210A
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- leaves
- puccinia
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- leaf powder
- diseased
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- 240000008042 Zea mays Species 0.000 title claims abstract description 41
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 26
- 241000221300 Puccinia Species 0.000 title claims abstract description 23
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 title claims abstract description 16
- 235000009973 maize Nutrition 0.000 title claims abstract description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 25
- 235000005822 corn Nutrition 0.000 claims abstract description 25
- 239000000843 powder Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000007605 air drying Methods 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 11
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 11
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 208000024891 symptom Diseases 0.000 claims description 4
- 239000002250 absorbent Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000009423 ventilation Methods 0.000 claims description 2
- 238000000399 optical microscopy Methods 0.000 claims 1
- 238000011081 inoculation Methods 0.000 abstract description 10
- 238000002360 preparation method Methods 0.000 abstract description 9
- 238000011160 research Methods 0.000 abstract description 7
- 239000000243 solution Substances 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract 3
- 238000007796 conventional method Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 241001123567 Puccinia sorghi Species 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229920000832 Cutin Polymers 0.000 description 2
- 241001415765 Polycarpa Species 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 244000000003 plant pathogen Species 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 230000028070 sporulation Effects 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 241000221301 Puccinia graminis Species 0.000 description 1
- 241001304534 Puccinia polysora Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Abstract
The invention provides a method for rapidly preparing a corn puccinia polycephala bacterial liquid in batches. The invention provides a method for rapidly preparing a maize puccinia polycistronica bacterial liquid in batches, which comprises the following steps: collecting diseased leaves caused by multiple piles of puccinia mairei, placing the diseased leaves in a cool and ventilated dry place for air drying for 2-6 days, selecting leaves without other sundry bacteria, crushing the selected leaves into leaf powder, dissolving the leaf powder in an aqueous solution containing a small amount of tween-80, filtering the aqueous solution through a double-layer medical gauze, washing filter residues for 3 times by using clear water and squeezing liquid in the filter residues completely, wherein the filtered solution is the prepared multiple piles of puccinia mairei bacterial liquid. The multi-stack puccinia strain liquid prepared by the method disclosed by the invention is high in speed and efficiency, can be used for preparing a large amount of bacteria liquid at one time, and is particularly suitable for situations such as field inoculation and the like requiring preparation of a large amount of bacteria liquid in a short time. The method provides efficient bacterial liquid preparation and inoculation method and technical support for the field research of the southern rust of the corn.
Description
Technical Field
The invention belongs to the field of plant disease research, and particularly provides a method for rapidly preparing a corn puccinia polycephala bacterial liquid in batches and application thereof.
Background
Southern corn rust is an air-borne disease caused by Puccinia polysora Underw, which occurs in many areas. In recent years, southern rust of corn has become one of the main diseases affecting corn production, and therefore, attention is increasingly paid to the research and control of corn puccinia polycarpa. The research object is gradually changed from a small amount of plants in the laboratory to a large amount of plants in the field, the research on the southern rust of the corn is carried out on the field scale, and the spore amount required by inoculation is far greater than that required by corresponding research in the laboratory. The conventional method for obtaining the spores is manually and visually picking, time and labor are wasted, the efficiency is low, and for a field test needing mass inoculation, the efficiency of obtaining the spores and preparing the bacterial liquid by the conventional method cannot meet the requirement of the field test, so that the southern corn rust test in a large range on the field scale is always limited by insufficient bacterial liquid for a long time.
Chinese patent 201510765142.1 discloses a single-spore propagation method of maize puccinia polycephala, which aims at obtaining a pure strain of maize puccinia polycephala. Chinese patent 201910852803.2 discloses an inoculation and propagation method of maize puccinia polycephala, which aims at performing rapid propagation on maize puccinia polycephala. Both methods cannot obtain a large amount of isolated spores in a short time and prepare a bacterial solution. Therefore, there is a need to develop a method for preparing a bacterial liquid of puccinia zeae with high efficiency and capable of being rapidly prepared in large quantities.
Disclosure of Invention
The puccinia polyclona produces a spore mass on the leaves of corn and retains the summer spores therein, which are cracked and released after complete maturation. Conventional methods for obtaining summer spores include shake-off, water washing, etc., which can only collect summer spores in cracked sporophytes, greatly limiting the number of spores obtained. The summer spores in the uncracked sporophyte also have infection capacity, and the conventional picking method (scraping by using an inoculating needle or an inoculating shovel) has low efficiency and is difficult to meet the requirement of quickly preparing a large amount of bacterial liquid.
In order to solve the problems, the inventor tries to improve the preparation method of the liquid of the maize puccinia polycistriformis, and finds that proper diseased leaves are dried and smashed, the summer spores in the uncracked sporophytes are fully exposed, and the water solution of tween-80 is used for spore extraction, so that a large amount of rapid extraction can be realized while the activity of the spores is kept.
On the one hand, the application provides a method for rapidly preparing a maize puccinia polycistriformis bacterial liquid in batches, which comprises the following steps: air drying the diseased leaves and crushing the diseased leaves to prepare leaf powder; dissolving the leaf powder in an aqueous solution containing tween-80; filtering; washing the filter residue with clear water.
Further, the air-drying the diseased leaves comprises placing the diseased leaves in a cool, ventilated and dry place for 2 to 6 days, preferably 3 to 5 days.
Further, the air-drying the diseased leaves comprises: flattening the blades, and spreading the blades on absorbent paper; air drying in shade and ventilation drying place for 5 days; during which the leaves were turned over every 12 hours.
Further, the crushing is to crush the air-dried diseased leaves by using an agricultural crusher to obtain corn leaf powder, and the diameter of the corn leaf powder is preferably less than 0.5 mm.
Further, the concentration of the aqueous solution of tween-80 is 0.01 to 0.03 percent, preferably 0.02 percent.
Further, the mass ratio of the corn leaf powder to the aqueous solution of the Tween-80 is 1:20-1:50, preferably 1: 30.
Further, the filtering is double-layer medical gauze filtering.
Further, the step of washing the filter residue with clear water is to wash the filter residue with clear water for 3 times, and the water consumption is less than 1/10 of the filtered bacterial liquid each time.
Further, the method comprises the steps of eliminating leaves and removing leaves containing other phytopathogens, preferably eliminating by disease symptom judgment and/or spore characteristic judgment through optical microscope observation.
On the other hand, the application provides the application of the method in the research on the southern rust of the corn and the control of the southern rust.
The diseased leaves are collected from the regions where the disease is naturally diseased in the field and the disease is serious (the area of the diseased regions accounts for more than 30% of the total area of the leaves), the diseased regions are relatively concentrated, and the leaves are clean and have no impurities.
Drawings
FIG. 1 is a photograph of air-dried corn leaves of example 1 of the present invention.
FIG. 2 is a photograph of pulverized leaf powder in example 1 of the present invention.
FIG. 3 is a picture of the disease manifestation after the inoculation of maize leaves in example 1 of the present invention.
FIG. 4 is a picture of the disease manifestation after the inoculation of maize leaves in comparative example 1 of the present invention.
FIG. 5 is a picture of the disease manifestation after the inoculation of maize leaves in comparative example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 method for rapidly preparing a liquid of puccinia polycistronica in batches
The method comprises the following steps of rapidly preparing the liquid of the puccinia polycephala in batches by the following method:
(1) collecting diseased leaves: collecting diseased leaves caused by infection of multiple puccinia zeae.
(2) Air drying the leaves: and (2) placing the diseased leaves collected in the step (1) in a cool and ventilated dry place for air drying for 5 days, flattening the leaves during air drying, spreading the leaves on absorbent paper, and turning the leaves once every 12 hours.
(3) Eliminating the blades: and (3) eliminating the air-dried leaves in the step (2) (shown in figure 1), removing the leaves containing other plant pathogens, and determining whether the leaves contain other plant pathogens by observing spore characteristics through an optical microscope.
(4) The leaves were crushed and dissolved in tween-80 water solution: and (3) crushing the leaves obtained in the step (3) by using an agricultural crusher to prepare leaf powder with the diameter of less than 0.5mm (shown in figure 2), and dissolving the leaf powder in 0.02% of Tween-80 aqueous solution, wherein the mass ratio of the leaf powder to the aqueous solution is 1: 30.
(5) Filtering the aqueous solution: and filtering the aqueous solution through double-layer medical gauze, washing the filter residue for 3 times by using clear water, completely squeezing the liquid in the filter residue, and obtaining the filtered solution, namely the prepared maize puccinia polycephala bacterial liquid.
(6) And (3) field inoculation: selecting healthy plants, wiping with sponge water to remove cutin on the surface of corn leaf, spraying the prepared bacterial liquid on the leaf with sprayer, and observing disease symptoms after 10 days (as shown in figure 3).
Taking two persons to treat the fresh diseased corn leaves with the weight of 5kg as an example, the preparation period of the bacterial liquid is 6.5 days, which are respectively as follows: leaf harvest for 1 day (8 hours of operation, the same below); air drying the leaves for 5 days; crushing the leaves for 1 hour; dissolving the leaf powder in water for 1 hour; filtration was carried out for 2 hours.
Comparative example 1 preparation of puccinia polycaprons bacterial liquid of corn by conventional method
The method comprises the following steps of preparing a corn puccinia polycarpa bacterial liquid, obtaining spores by adopting a traditional inoculating needle picking method, and preparing the bacterial liquid:
(1) collecting diseased leaves: collecting diseased leaves caused by infection of multiple puccinia zeae.
(2) Collecting maize puccinia polyclona spores: determining the disease incidence area by adopting an artificial visual mode, picking (scraping) summer spores of disease incidence leaves in a sporulation area by using an inoculating needle (inoculating shovel), and placing the obtained spores in a clean and dry container (such as a centrifuge tube and the like).
(3) Spore suspension preparation: the collected spores were dissolved in 0.02% aqueous tween-80 solution and stirred well.
(4) And (3) field inoculation: selecting healthy plants, wiping with sponge to remove cutin on the surface of corn leaf, spraying the prepared bacterial liquid on the leaf with sprayer (or small spray pot), and observing disease symptoms after 10 days (as shown in figure 4). Compared with the figure 3, the bacterial liquid prepared by the two methods can be sprayed to ensure that the leaves normally attack, and other diseases cannot be caused.
Similarly, taking the two persons to treat the diseased corn leaves with fresh weight of 5kg as an example, the preparation period of the bacterial liquid is about 13 days, which are respectively as follows: collecting leaves for 1 day; picking multiple piles of puccinia graminis spores of corn for 12 days; spore suspensions were prepared for 1 hour.
Compared with the bacterial liquid preparation efficiency of the conventional method, when the fresh weight of the diseased leaves to be treated is 5kg, the bacterial liquid preparation efficiency of the method is 2 times that of the conventional method, and when more leaves to be treated exist, the efficiency advantage of the method is more obvious. In addition, the manual workload required by the invention is greatly reduced.
Example 3 spore utilization was higher and lower compared to the present and conventional methods
The leaves after sporulation according to the method of comparative example 1 were air-dried, then inoculated on healthy leaves in the field after the same procedure as in example 1 (crushing, dissolving in water, filtering, washing), and found to be normal in disease incidence (as shown in fig. 5). The method for manually picking the spores is proved to inevitably leave out the spores, so that the utilization rate of the spores is reduced.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for rapidly preparing a maize puccinia polycistriformis bacterial liquid in batches is characterized by comprising the following steps: air drying the diseased leaves and crushing the diseased leaves to prepare leaf powder; dissolving the leaf powder in an aqueous solution containing tween-80; filtering; washing the filter residue with clear water.
2. The method of claim 1, wherein air drying diseased leaves comprises: the diseased leaf is placed in a cool, ventilated and dry place for 2-6 days, preferably 3-5 days.
3. The method of claim 2, wherein air drying diseased leaves comprises: flattening the blades, and spreading the blades on absorbent paper; air drying in shade and ventilation drying place for 5 days; during which the leaves were turned over every 12 hours.
4. The method according to any one of claims 1 to 3, wherein the crushing is carried out by crushing the air-dried diseased leaves with an agricultural crusher to obtain the corn leaf powder, preferably the corn leaf powder has a diameter of less than 0.5 mm.
5. The method according to any one of claims 1-4, wherein the aqueous solution of tween-80 is at a concentration of 0.01 to 0.03%, preferably 0.02%.
6. The method according to claim 5, wherein the mass ratio of the corn leaf powder to the aqueous solution of tween-80 is 1:20 to 1:50, preferably 1: 30.
7. The method of any one of claims 1-6, wherein the filtration is a double layer medical gauze filtration.
8. A method according to any one of claims 1 to 7 wherein washing the cake with clear water is carried out 3 times with clear water, each time with less than 1/10 of the filtered liquid.
9. The method according to any one of claims 1 to 8, wherein the method further comprises the step of culling the leaves, removing leaves containing other phytopathogens, preferably by disease symptom judgment and/or spore characteristic judgment by optical microscopy.
10. Use of the method according to any one of claims 1 to 9 in the study of southern rust in maize and the control thereof.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565688A (en) * | 2008-04-25 | 2009-10-28 | 中国科学院沈阳应用生态研究所 | Trichodrema spp. microbial agent and preparation method thereof |
| CN103333851A (en) * | 2013-07-09 | 2013-10-02 | 湖南泰谷生物科技股份有限公司 | Method of producing conidiospore by solid fermentation of trichoderma harzianum |
| US20180155412A1 (en) * | 2015-05-13 | 2018-06-07 | The Trustees Of The University Of Pennsylvania | Aav-mediated expression of anti-influenza antibodies and methods of use thereof |
| CN110551674A (en) * | 2019-09-10 | 2019-12-10 | 中国农业大学 | Inoculation and propagation method of maize puccinia polycarya |
-
2021
- 2021-12-27 CN CN202111612546.9A patent/CN114214210A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565688A (en) * | 2008-04-25 | 2009-10-28 | 中国科学院沈阳应用生态研究所 | Trichodrema spp. microbial agent and preparation method thereof |
| CN103333851A (en) * | 2013-07-09 | 2013-10-02 | 湖南泰谷生物科技股份有限公司 | Method of producing conidiospore by solid fermentation of trichoderma harzianum |
| US20180155412A1 (en) * | 2015-05-13 | 2018-06-07 | The Trustees Of The University Of Pennsylvania | Aav-mediated expression of anti-influenza antibodies and methods of use thereof |
| CN110551674A (en) * | 2019-09-10 | 2019-12-10 | 中国农业大学 | Inoculation and propagation method of maize puccinia polycarya |
Non-Patent Citations (4)
| Title |
|---|
| 李石初等: "玉米杂交种对广西玉米主要病害抗性鉴定", 东北农业科学, vol. 41, no. 2, pages 2 - 1 * |
| 王淼等编: "食品风味物质与生物技术", 31 May 2004, 北京：中国轻工业出版社, pages: 276 * |
| 石菁等: "玉米瘤黑粉病抗性鉴定技术的评价", 玉米科学, vol. 18, no. 1, pages 1 * |
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