CN111235034B - Method for distinguishing southern rust of corn from common rust spore - Google Patents
Method for distinguishing southern rust of corn from common rust spore Download PDFInfo
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- 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
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
Abstract
The invention discloses a method for distinguishing southern rust of corn from common rust spores, belonging to the technical field of plant pathology. The method comprises the following steps: the common rust of corn and the southern rust spore are taken to be processed on a glass slide carrying hydrochloric acid or sulfuric acid solution and immediately observed under a microscope. The results show that protoplasts of southern rust fungus spores treated by hydrochloric acid are concentrated into a round large cluster, while protoplasts of common rust fungus are concentrated into a plurality of small clusters; after the sulfuric acid treatment, the protoplast layer of the southern rust fungus appears as a large cluster, while the protoplast of the common rust fungus appears as a plurality of small clusters. The method is simple and convenient to operate, can be realized under the conditions of laboratories, greenhouses and the like, is convenient to observe, is convenient and easy to obtain equipment and medicament, and greatly saves the experiment and time cost.
Description
Technical Field
The invention belongs to the technical field of plant pathology, and particularly relates to a method for distinguishing southern rust of corn from common rust spores.
Background
The corn rust diseases in China mainly include common rust diseases and southern rust diseases. Common rust disease (Common corn rust) of corn and Southern rust disease (Southern rust) of corn caused by Puccinia polysora Underw are serious diseases prevalent in China.
Common corn rust pathogenic bacteria summer spores are nearly spherical and elliptical, have the sizes of 23-32 Mumx (20-31) Mum, are light yellow or tawny, and have thorns on the surfaces. The southern rust fungus summer spores of the corn are round or elliptical and have single cells, a small amount of spiny protrusions are arranged on the surface, the southern rust fungus summer spores are orange to red brown, the southern rust fungus summer spores are 22.5-35 mu m multiplied by 26.7-44 mu m, and the average southern rust fungus summer spores are 25.71 mu m multiplied by 33.46 mu m. Under the field condition, due to obvious climate difference in different areas, various corn varieties and the like, the corn rust types cannot be distinguished obviously, the corn rust mainly damages the upper part of the soil surface, and the corn rust can infect fruit clusters, bracts and even male flowers when serious. In the early stage of the disease, the sporangium of yellow spots are gathered or scattered at the base, the upper part and two sides of the leaf to form scattered or gathered yellow sporophyte of summer sporangium. When the corn rust is serious, leaves are covered with spore heaps, photosynthesis is influenced, the leaves are dry, seeds are not full, and yield is reduced or even the seeds are not harvested completely. Common rust and southern rust frequently occur together in southwest areas and northern areas, and cross contamination is very easy to identify in the mixed areas in experimental research, so that the breeding and research work is seriously hindered. However, the sources and varieties are not easily distinguished after greenhouse cultivation, macroscopic morphology is similar (shown in fig. 1 and fig. 2), and bacterial source pollution is caused, so that work is useless. In the prior art, the southern rust of corn and the common rust of corn can be distinguished by adopting a Realtime-PCR molecular means, or the ITS sequence is utilized to carry out molecular detection on the corn rust in China. However, the identification process is complicated, the requirements on equipment and medicament are high, and the experiment cost and the time cost are high. In the experiment, a rapid identification method is urgently needed to identify the types of the corn rust in the fields and greenhouses.
Disclosure of Invention
Aiming at the problems, the invention provides a method for distinguishing southern rust of corn from common rust spores, which comprises the following steps:
putting the spores of the rust fungi on a glass slide loaded with a hydrochloric acid or sulfuric acid solution, covering a cover glass, and observing and distinguishing under a microscope;
the distinguishing method comprises the following steps:
after the treatment of the hydrochloric acid solution, if the protoplasts of the rust spores are concentrated into a round big ball, the rust is southern rust, and if the protoplasts of the rust spores are concentrated into a plurality of small balls or are unchanged, the rust is common rust;
after the treatment of the sulfuric acid solution, if a large colony appears in the protoplast of the rust fungus spore, the protoplast is southern rust fungus; if the protoplast of the rust spore has multiple small masses, the rust spore is common rust
The concentration of the sulfuric acid is 13.2-13.8 mol L-1(ii) a The concentration of hydrochloric acid is 9-12 mol L-1。
The concentration of the sulfuric acid is 13.8 or 13.2mol L-1The concentration of hydrochloric acid is 11.4 or 9mol L-1。
The method for distinguishing southern corn rust from common rust spores also includes the cultivation of rust spores.
The method for cultivating the rust fungus spores comprises the following steps: the spores of southern rust or common rust of corn are picked up or sprayed on healthy corn seedlings.
The healthy corn seedlings are corn seedlings in a three-leaf one-heart seedling stage; the inoculated leaf is on the second leaf and/or the third leaf of the healthy corn seedling.
Before inoculating healthy corn seedlings, carrying out dewaxing treatment on bacterial leaves, and treating the dewaxed leaves by using a Tween 20 aqueous solution with the mass fraction of 0.05%.
After the healthy corn seedlings are inoculated with the bacteria, the temperature is kept at 20-30 ℃, the relative humidity of air is more than or equal to 80%, under the condition, the healthy corn seedlings are firstly cultured in a natural light environment for 24-48h, and then are continuously cultured in an environment with sufficient illumination.
And inoculating the spores for 1-2 generations.
The invention has the beneficial effects that:
1. according to the method, when the diluted hydrochloric acid is used for treating maize southern rust grass spores, protoplasts of the maize southern rust grass spores are concentrated into a round large cluster, while the protoplasts of common maize rust are not concentrated into a plurality of protoplasts with different sizes. The diluted sulfuric acid treated corn southern rust protoplast has one large colony, while the corn common rust protoplast has a plurality of small colonies, so the operation is simple and convenient, the southern rust protoplast can be realized under the conditions of laboratories, greenhouses and the like, the observation is convenient, and the distinguishing degree is large.
2. The experimental method has the advantages of simple and convenient identification process, convenient and easily-obtained equipment and medicament, and greatly saves the experimental cost and the time cost.
Drawings
FIG. 1 is a morphological diagram of greenhouse maize Puccinia communis;
FIG. 2 is a morphogram of southern rust of greenhouse maize;
FIG. 3 is a morphological diagram of common rust spores of corn after being treated with clear water;
FIG. 4 is a morphological diagram of southern rust spores of maize after treatment with clear water;
FIG. 5 is 95:25(HCl: H)2O) a morphogram of a maize common rust spore in proportion;
FIG. 6 is 95:25(HCl: H)2O) a morphogram of southern rust spores of corn;
FIG. 7 shows 72:28 (H)2SO4:H2O) a morphogram of a maize common rust spore in proportion;
FIG. 8 shows 72:28 (H)2SO4:H2O) ratio of southern rust spores of maize.
Detailed Description
The invention is described in further detail below with reference to the following figures and specific examples:
as shown in figures 1 and 2, the rust fungus of corn has fast sporulation on leaves, has no unbroken sporophyte in the field in shape, is yellow brown or dark brown and is not easy to distinguish (figures 1 and 2). Common rust spores of corn are mostly dark brown and like to infect the back of leaves, and southern rust spores of corn are yellowish brown and rust-shaped and can infect the back or stem of leaves in a small amount. However, the spore forms of the two bacteria are not unique and cannot be distinguished perfectly.
1) Seedling; comprises accelerating germination and sowing of corn seeds; the germination accelerating method comprises the following steps: selecting plump corn seeds, wherein the corn varieties are Tiangui glutinous 932 and Zhengdan 95. Soaking the seeds for 24-48h at room temperature with 0.1% hydrogen peroxide, taking out the seeds, covering the seeds with wet paper towels or wet cloths, and accelerating germination at ventilated places. The corn seeds can be sown after 1-2mm radicles grow at the front ends of the corn seeds, and during sowing, the radicles face upwards and face to the center of the planting container, and the seeds are circular along the periphery of the pot, so that bacteria receiving and collecting are facilitated.
2) Initial breeding of strains: selecting a single spore pile of southern rust and common rust of corn on diseased leaves, inoculating the single spore pile on a second leaf and/or a third leaf of a healthy corn seedling, and inoculating only one single spore pile on each leaf; the strain used for southern rust of corn is sampled in the south of the 3 th month of 2019 and the south of the 9 th month of 2019. The test strains are obtained by the propagation of Hainan subculture living bodies and the propagation of Henan Kaifeng separated living bodies, the common rust of the corn is obtained from 9-month Ningxia primordium sampling in 2019 and naturally occurs in 10-month greenhouse in 2019.
3) Bacterial strain transfer: after the early-bred maize seedlings are diseased, spores of southern rust and common rust of the maize growing on the leaves are picked or sprayed on second leaves and third leaves of a plurality of healthy maize seedlings.
Step 2) and 3) the healthy corn seedlings are corn seedlings in a three-leaf one-heart seedling stage; before inoculating healthy corn seedlings, dewaxing second leaves and third leaves of the corn seedlings, and treating the dewaxed leaves by using a Tween 20 aqueous solution with the mass fraction of 0.05%. The corn seedlings can be transplanted for 1-2 generations. After inoculation, the temperature is kept at 20-30 ℃, the moisture is kept for 24-48h, then the culture is continued under the condition of sufficient illumination, and the relative humidity of air is kept to be more than or equal to 80 percent and the temperature is kept at 20-30 ℃.
4) And (3) inoculating single spores of southern rust and common rust of corn on leaves of healthy corn seedlings in a stacking manner, and propagating sufficient spores through transfer culture.
Examples
1) Soaking seeds of Tiangui glutinous 932 or Zhengdan 958 corn for 24 hours, moisturizing and accelerating germination with gauze for 33 hours, then sowing in a pot, mixing soil with turf and vermiculite one by one, then potting, sowing eight corn seeds in one pot near the periphery of the pot, and after watering, under the condition of 30 ℃, growing into three-leaf and one-heart healthy seedlings after fifteen days. And 3, in 2019, sampling south China sea in the south China, and carrying out subculture propagation until inoculation. Firstly, dewaxing the corn leaves, so that the Tween-20 solution can be attached to the leaves, and the corn rust fungi can be better infected. If the corn leaves do not remove the wax and wash the seedlings, the Tween-20 solution is easily gathered into water drops to fall down, and the inoculation effect is greatly reduced.
2) After preparing a healthy corn seedling for inoculation, selecting a spore pile of southern rust of corn on a diseased leaf, spraying 0.50% Tween-20 solution on the corn seedling, uniformly coating spore powder on a second leaf and a third leaf, transferring and spraying new healthy corn seedlings to the corn seedling with a three-leaf one-heart seedling stage after propagation, preserving moisture for 24-48h under the condition of natural illumination at 20-30 ℃, and then transferring to the environment with sufficient illumination, relative air humidity of more than 80% and temperature of 20-30 ℃ for continuous culture for fifteen days to obtain fresh spores. At present, the southern rust of corn can not be preserved except for the living subculture propagation preservation. The Hainan strain is inoculated repeatedly and cultured for ten months. Collecting spore powder after spores of all strains are basically enough. Adding 0.50% Tween-20 into the collected spore powder to prepare spore suspension, spraying and inoculating on multiple healthy corn seedlings, and propagating to a sufficient amount.
3) And breeding the required using amount of the southern rust fungus and the common rust fungus of the corn.
4) Sulfuric acid with the mass fraction of 98 percent is diluted into H2SO4:H2O72: 28, 73:27, 74:26, 75: 25; hydrochloric acid with the mass fraction of 36-38% is diluted with HCl to H2Aqueous solutions of sulfuric acid and hydrochloric acid were prepared at 75:25, 95:5, and 100: 0. The protoplast changes of the common rust fungus of corn and the southern rust fungus of corn are compared after the common rust fungus and the southern rust fungus of corn are respectively treated by diluted sulfate acid. Firstly, respectively picking a small amount of fresh common rust of corn and southern rust of corn on a glass slide carrying a sulfuric acid solution by using a needle, covering the glass slide with a cover glass, and immediately placing the glass slide under a microscope for observation. A small amount of common rust of corn and southern rust of corn spores are picked by a needle and put on a glass slide carrying a hydrochloric acid solution, and then a cover glass is covered and immediately placed under a microscope for observation.
The statistics of the morphology after hydrochloric acid treatment at different concentrations, as shown in Table 1, for more than 50 spores selected per experiment, are shown in volume ratio (HCl: H)2O) concentration of southern rust protoplasts of maize at a ratio of 75:25 toLarge clumps, while common rust of corn did not change.
When volume ratio (HCl: H)2O)95:5, the protoplasts of the maize rust fungus are obviously different, the protoplasts of the maize southern rust fungus spores are concentrated into a large group in the visual field (figure 6), and the protoplasts of the maize common rust fungus are not concentrated into a plurality of small groups (figure 5) under the same proportion.
When volume ratio (HCl: H)2O) to 100: southern rust of maize (0) concentrates large clumps while common rust of maize does not appear as one or several large clumps.
TABLE 1 variation of spore protoplasts under hydrochloric acid treatment of different concentrations
In Table 1, PBHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, PSHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, NBHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, and NSHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize.
The statistics of spore morphology after sulfuric acid treatment at different proportional concentrations are shown in Table 2, at volume ratio (H)2SO4:H2O)72:28 later, the protoplasts showed differences in the ratio, with 98% of the spore protoplasts from southern Ruscus zeae concentrated to one large clump in the field of view (FIG. 8), and with the same ratio of common Ruscus zeae, the protoplasts were not concentrated to multiple small clumps (FIG. 7). In volume ratio (H)2SO4:H2O)72:28 to 75:25, the shape of the large lumps tends to be rounded, assuming an irregularly dispersed state.
TABLE 2 Change in spore protoplasts with different concentrations of sulfuric acid treatment
In Table 2, PBH2SO4: number of large clusters (n: more) of common rust spores of maize after sulfuric acid treatmentPSH), PSH2SO4: number of colonies (n: more) after sulfuric acid treatment of maize common rust spores, NBH2SO4: NSH (number of clusters) of southern rust spores of maize treated with sulfuric acid2SO4: number of colonies (n: more) after sulfuric acid treatment of southern rust spores of maize.
Comparative example 1
Steps 1) -3) are the same as in example 1.
A small amount of common rust of corn and southern rust of corn spores were picked with a needle onto a glass slide with distilled water, covered with a cover glass, and immediately placed under a microscope for observation.
The results of direct observation of the characteristics of spores of common rust of maize and southern rust of maize (figures 3 and 4) after the spores of common rust of maize and southern rust of maize are treated by distilled water show that the separation of plasmolysis of common rust of maize and southern rust of maize does not occur. Common rust of corn is mostly round, a few elliptical monospores, southern rust of corn is mostly elliptical, and a few circular monospores. The bacteria with similar shapes can not be clearly distinguished, and the surfaces of the bacteria have spine-shaped protrusions. In addition to typical symptoms, indistinguishable conditions may also occur. The color difference is not large, the morphology is close, and the color difference cannot be distinguished by a microscope. Changes in the protoplasts of maize common rust and maize southern rust spores under distilled water treatment are shown in table 3.
TABLE 3 Change in spore protoplasts of Puccinia zeae and southern Ruscus zeae under distilled water treatment
In Table 3, PBH2O: protoplast mass number, PSH, of corn common rust treated with clear water2O: small clump number, NBH, of protoplasts after treatment of puccinia fusca with clear water2O: number of protoplast boluses, NSH, after southern corn rust is treated by clear water2O: number of colonies of protoplasts after treatment of southern rust on maize with clear water.
It is shown that the corn rust fungi have different shapes, oval or round shapes due to different sources.
Comparative example 2
Steps 1) -3) are the same as in example 1.
4) Hydrochloric acid with the mass fraction of 36-38% is diluted with HCl to H2Aqueous solutions of sulfuric acid and hydrochloric acid were prepared at 0:100, 35:65, and 55: 45. The protoplast change of the common rust fungus of corn and the southern rust fungus of corn is compared after the common rust fungus and the southern rust fungus of corn are respectively treated by diluted hydrochloric acid. A small amount of common rust of corn and southern rust of corn spores are picked by a needle and put on a glass slide carrying a hydrochloric acid solution, and then a cover glass is covered and immediately placed under a microscope for observation.
The morphological statistics of more than 50 spores selected for each experiment after treatment with hydrochloric acid at different concentrations are shown in Table 4, at a volume to volume ratio of t (HCl: H)2O)0:100, 35:65, 55:45, no significant change in protoplasts occurred.
TABLE 4 variation of spore protoplasts under hydrochloric acid treatment of different concentrations
In Table 4, PBHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, PSHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, NBHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize, and NSHCl represents the number of colonies after hydrochloric acid treatment of southern rust of maize.
Comparative example 3
4) Sulfuric acid with the mass fraction of 98 percent is diluted into H2SO4:H2O ═ 0:1, 35:65, 55:45, 95:5, 100: 0. the common rust fungus of corn and the southern rust fungus of corn are respectively treated by diluted sulfuric acid and then the protoplast change of the two fungus is compared. Firstly, respectively picking a small amount of fresh common rust of corn and summer spores of southern rust of corn on a glass slide loaded with a sulfuric acid solution by using a needle, covering the glass slide, and immediately placing the glass slide under a microscope for observation.
The statistics of spore morphology after sulfuric acid treatment at different proportional concentrations are shown in Table 5, at volume ratio (H)2SO4:H2O)0:1, 35:65, 55:45, no change in protoplasts; when volume ratio (H)2SO4:H2O)95:5, 100: at 0, there was no microscopic observable difference between the two.
TABLE 5 Change in spore protoplasts with different concentrations of sulfuric acid treatment
In Table 5, PBH2SO4: the number of large clusters (n: more) of the common rust fungus spores of the corn after being treated by sulfuric acid, PSH2SO4: number of colonies (n: more) after sulfuric acid treatment of maize common rust spores, NBH2SO4: NSH (number of clusters) of southern rust spores of maize treated with sulfuric acid2SO4: number of colonies (n: more) after sulfuric acid treatment of southern rust spores of maize.
The test strains have different spore forms due to different factors such as corn varieties, regions and the like, but the protoplast changes in the experiment are more consistent. There may be some shape differences between the different sources of the fungus, but the southern corn rust and common corn rust differ in their morphology as a function of acid concentration when examined microscopically. The method can be used for conveniently and quickly carrying out auxiliary identification on the corn rust types, has the advantages of small using amount of spores, low cost and simple and convenient operation, is convenient for field agricultural science and technology workers to master, and is applied to identification of field corn rust and identification in the research of breeding bacteria. However, in scientific research experiments, molecular experiments are more accurate and sensitive if time and experimental equipment allow. The protoplast change phenomenon of the corn rust fungus spores after being treated by hydrochloric acid or sulfuric acid solution is not the only means for identifying common rust fungus and southern rust fungus of corn, and the protoplast change phenomenon is primarily judged by combining the morbidity situation, the color and shape of a spore pile and the like in the application and then comprehensively judged by combining acid treatment and molecular means.
Claims (8)
1. A method for distinguishing southern corn rust from common rust spores is characterized by comprising the following steps:
putting the spores of the rust fungi on a glass slide loaded with a hydrochloric acid or sulfuric acid solution, covering a cover glass, and observing and distinguishing under a microscope;
the distinguishing method comprises the following steps:
after the treatment of the hydrochloric acid solution, if the protoplasts of the rust spores are concentrated into a round big ball, the rust is southern rust, and if the protoplasts of the rust spores are concentrated into a plurality of small balls or are unchanged, the rust is common rust;
after the treatment of the sulfuric acid solution, if a large colony appears in the protoplast of the rust fungus spore, the protoplast is southern rust fungus; if the protoplast of the rust fungus spore has a plurality of small groups, the rust fungus is common rust fungus;
the concentration of the sulfuric acid solution is 13.2-13.8 mol.L-1(ii) a The concentration of the hydrochloric acid solution is 9-12 mol.L-1。
2. The method of claim 1, wherein the sulfuric acid solution has a concentration of 13.8 or 13.2 mol-L-1The concentration of the hydrochloric acid solution is 11.4 or 9 mol.L-1。
3. The method of any one of claims 1-2, further comprising culturing the rust spores.
4. The method of claim 3, wherein the method of culturing the rust spores comprises: the spores of southern rust or common rust of corn are picked up or sprayed on healthy corn seedlings.
5. The method of claim 4, wherein the healthy corn seedlings are corn seedlings at the trilobate single-heart seedling stage; the inoculated leaf is on the second leaf and/or the third leaf of the healthy corn seedling.
6. The method of claim 4, wherein before the healthy corn seedlings are inoculated, the leaves of the healthy corn seedlings are dewaxed, and the dewaxed leaves are treated with a 0.05% tween 20 aqueous solution.
7. The method as claimed in claim 4, wherein after inoculation of the healthy maize seedlings, the temperature is kept at 20-30 ℃, the relative humidity of air is more than or equal to 80%, under the condition, the healthy maize seedlings are firstly cultured in a natural light environment for 24-48h, and then the healthy maize seedlings are moved to an environment with sufficient illumination for continuous culture.
8. The method of claim 4, wherein the spores are transferred for 1-2 generations.
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