CN110029083B - Method for separating plasmodiophora brassicae monospores based on methylene blue agarose method - Google Patents

Abstract

The invention provides a method for separating plasmodiophora brassicae monospores based on a methylene blue agarose method, which comprises the following steps of firstly preparing 5 × 10³Spore. mL^‑1The plasmodiophora brassicae dormant spore suspension; adding 0.01% methylene blue staining solution into 1.3% agarose solution to prepare a methylene blue agarose slide gel layer; uniformly coating 2 mu L of plasmodiophora brassicae resting spore suspension on the surface of a gel layer, observing under a microscope, separating the gel layer containing monospores when only one spore is determined in the visual field, inversely covering the gel layer on the root hair of a crucifer seedling, and culturing to obtain the plasmodiophora brassicae monospores. The method disclosed by the invention is used for separating the plasmodiophora brassicae monospores by a methylene blue agarose method, the background is slightly blue, and no impurities or air bubbles exist, so that the plasmodiophora brassicae monospores can be clearly distinguished. The method has simple process and easy operation, and greatly improves the efficiency and the accuracy of the single-spore separation of the plasmodiophora brassicae.

Description

Translated fromChinese

一种基于亚甲基蓝琼脂糖法分离芸薹根肿菌单孢的方法A kind of method based on methylene blue agarose method to separate P. brassicae monospores

技术领域technical field

本发明属于涉及单孢分离领域，具体涉及一种基于亚甲基蓝染色液结合琼脂糖法的芸薹根肿菌单孢分离方法。The invention belongs to the field of single spore separation, in particular to a single spore separation method of P. brassicae based on methylene blue staining solution combined with agarose method.

背景技术Background technique

十字花科根肿病(Clubroot disease)是由芸薹根肿菌(Plasmodiophorabrassicae Woron.)侵染引起的一种专性寄生的世界性病害，素有“十字花科癌症”之称，任何十字花科植物的感病品种都能被侵染，引起薄壁细胞的异常分裂增大，形成瘤状肿根。根肿菌休眠孢子在无感病寄主植物的土壤中存活可达20年之久，土壤一旦污染，将不再适宜十字花科植物的栽培。近年来，根肿病在我国各地区迅速蔓延，其中西南、东北、华东等地区成为根肿病的重灾区。据《中国农业年鉴》统计，由根肿病危害造成的十字花科作物经济损失超过40亿元，在产业链的前、中、后连带影响，造成经济损失超过100亿元。Cruciferous clubroot disease (Clubroot disease) is an obligate parasitic worldwide disease caused by Plasmodiophorabrassicae Woron. The susceptible varieties of plants of the family can be infected, causing abnormal division and enlargement of parenchyma cells, forming tumor-like tumor roots. The dormant spores of P. rhizogenes can survive for up to 20 years in the soil of non-susceptible host plants. Once the soil is polluted, it will no longer be suitable for the cultivation of cruciferous plants. In recent years, clubroot has spread rapidly in various regions of my country, among which Southwest, Northeast, East China and other regions have become the hardest hit areas of clubroot. According to the "China Agricultural Yearbook" statistics, the economic loss of cruciferous crops caused by clubroot disease exceeds 4 billion yuan, and the economic loss in the front, middle and rear of the industrial chain is more than 10 billion yuan.

选用抗病品种是防治十字花科根肿病的有效途径。由于芸薹根肿菌种下存在生理小种，生产中经常出现十字花科寄主品种对病原菌某个或少数生理小种免疫或高抗，而对另一些生理小种则高度感染的现象。由于抗病性是针对某个或某些生理小种的，生产上大面积单一地推广具有该类抗性的品种时，容易导致侵染它的生理小种上升为优势小种而被感染、淘汰，因而抗病性不能稳定和持久。获得纯化的芸薹根肿菌单孢系是开展生理小种鉴定、病原菌遗传变异，以及稳定、持久抗病育种等许多研究工作的基础。Selection of disease-resistant varieties is an effective way to prevent and treat cruciferous clubroot. Due to the existence of physiological races under the species of Rhizoctonia brassicae, it often occurs that the host species of Cruciferae are immune or highly resistant to one or a few physiological races of pathogens, while they are highly infected with other physiological races. Since the disease resistance is aimed at one or some physiological races, when a variety with this type of resistance is promoted in a large area in production, it is easy to cause the physiological race that infects it to become a dominant race and be infected, culling, so disease resistance is not stable and durable. Obtaining the purified P. Brassica monospore line is the basis for many researches such as identification of physiological races, genetic variation of pathogenic bacteria, and stable and lasting disease resistance breeding.

芸薹根肿菌属于严格的专性寄生菌，不能进行人工分离培养，只能从活体宿主植株中吸收营养而繁殖，离开宿主生物就不能继续生存，因此很难分离获得纯菌株单孢系。P. brassicae is a strict obligate parasite, which cannot be artificially isolated and cultured. It can only absorb nutrients from living host plants and reproduce. It cannot continue to survive without the host organisms. Therefore, it is difficult to isolate and obtain pure strains of monospores.

目前国内外根肿菌单孢分离的方法主要有以下四种：At present, there are four main methods for the isolation of P. rhizogenes at home and abroad:

第一种方法是水琼脂块法分离根肿菌单孢子，将孢悬液滴于含有水琼脂块的载玻片上，在光学显微镜物镜转换器上安置微型打孔器准确切取一个孢子放于寄主根部。这种方法存在以下问题：水琼脂块制备过程中易产生气泡，而且含有杂质较多，而芸薹根肿菌休眠孢子无色、非常小，单孢大小4.6～6.0×1.6～4.6μm，水琼脂块在显微镜下观察很难区分孢子、气泡或杂质，往往不能准确判断是否含有芸薹根肿菌单孢，导致切取的单孢目标块不含或含有多个芸薹根肿菌单孢。The first method is to separate the single spore of P. rhizogenes by the water agar block method, drop the spore suspension on the glass slide containing the water agar block, and place a micro puncher on the optical microscope nosepiece to accurately cut out a spore and place it on the host. root. This method has the following problems: bubbles are easily generated during the preparation of water agar blocks, and there are many impurities, while the dormant spores of P. brassicae are colorless and very small, with a single spore size of 4.6-6.0 × 1.6-4.6 μm, and the water It is difficult to distinguish spores, air bubbles or impurities from the agar block under the microscope, and it is often impossible to accurately determine whether it contains a single spore of P.

第二种方法是微管法(李茜等，2012，芸薹根肿菌(Plasmodiophora brassicae)单孢分离接种及生理小种的鉴定.植物保护，2012，38(3):95-101)，借助微管在水琼脂块上准确吸取一个孢子放于寄主根部。该方法除存在水琼脂块气泡和杂质多，不易区分芸薹根肿菌单孢的问题外，还存在利用微管反复吸取悬浮液时易出现孢子沾在微管壁上导致使观察不到的问题，该方法效率较低。The second method is the microtubule method (Li Qian et al., 2012, Isolation and inoculation of single spores of Plasmodiophora brassicae and identification of physiological races. Plant Protection, 2012, 38(3): 95-101), A single spore was accurately picked up on a water agar block with the aid of a micropipette and placed on the root of the host. In addition to the problem that the water agar block has many bubbles and impurities, and it is difficult to distinguish single spores of P. brassicae, the method also has the problem that the spores are easily stuck on the wall of the microtubule when the suspension is repeatedly sucked by the microtubule, which makes the observation invisible. The problem is that this method is less efficient.

第三种方法是冷冻盒接菌法(刘一凡等，2017，芸薹根肿菌单孢分离技术体系构建及沈阳地区根肿菌的鉴定.园艺学报，2017，44(12):2383-2390)，借助针灸针在水琼脂块上准确挑取一个孢子准确放于寄主根部，培养于81孔冷冻盒。该方法也是在传统水琼脂块上在光学显微镜下利用针灸针挑取一个单孢，同样存在挑取目标孢子可能是气泡或杂质的问题，此外在挑取过程中针灸针会使孢子壁破碎，直接导致孢子死亡，影响发病率，同时该方法在挑取单孢时耗时较久。The third method is the frozen box inoculation method (Liu Yifan et al., 2017, Construction of a single spore isolation technology system for Plasmorhizium Brassica and identification of P. , with the help of acupuncture needles, accurately pick a spore on the water agar block and place it on the root of the host, and cultivate it in an 81-well freezer box. This method also uses acupuncture needles to pick a single spore on a traditional water agar block under an optical microscope. There is also the problem that the picked target spores may be air bubbles or impurities. It directly leads to the death of spores and affects the morbidity rate. At the same time, this method takes a long time to pick single spores.

第四种方法是通过稀释芸薹根肿菌休眠孢子悬浮液，使悬浮液达到1μL含有一个休眠孢子的浓度，然后用移液枪在载玻片上进行反复观察，确定只有一个孢子后进行接种。该方法由于休眠孢子悬液中孢子分布不均匀，可能接种的单孢悬浮液不含有孢子或不是单孢。The fourth method is to dilute the suspension of P. brassicae dormant spores to a concentration of 1 μL containing one dormant spore, and then repeatedly observe on the slide with a pipette to confirm that there is only one spore before inoculation. Due to the uneven distribution of spores in the dormant spore suspension in this method, it is possible that the inoculated single spore suspension does not contain spores or is not a single spore.

以上4种现有技术均存在琼脂块中存在气泡或杂质，孢子与背景差异不明显、不易区分单孢等问题，导致挑取单孢耗时、费力，成功率较低。The above four existing technologies all have problems such as the existence of air bubbles or impurities in the agar block, the inconspicuous difference between spores and the background, and the difficulty in distinguishing single spores, resulting in time-consuming and laborious picking of single spores, and a low success rate.

发明内容SUMMARY OF THE INVENTION

为解决现有技术存在的上述问题，本发明的目的在于提供一种基于亚甲基蓝染色液与琼脂糖法相结合的芸薹根肿菌单孢分离方法。用琼脂糖法替代水琼脂法，能有效避免水琼脂中有气泡或杂质干扰的问题；在琼脂糖中加入亚甲基蓝染色液，琼脂糖背景呈现轻微蓝色，而芸薹根肿菌休眠孢子无色，更易于区分单孢和琼脂糖背景，解决了芸薹根肿菌单孢目标模糊、不易挑取等缺点。本发明操作简单，能大大提高挑取单孢的准确性，提高了接种后获得单孢系的可能性。In order to solve the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a method for separating single spores of P. brassicae based on the combination of methylene blue staining solution and agarose method. Using the agarose method instead of the water agar method can effectively avoid the problem of air bubbles or impurities in the water agar; adding methylene blue staining solution to the agarose, the background of the agarose appears slightly blue, while the resting spores of P. brassicae are colorless , it is easier to distinguish single spores from agarose background, and solves the shortcomings of P. The invention has simple operation, can greatly improve the accuracy of picking single spores, and improves the possibility of obtaining a single spore line after inoculation.

本发明所提供的基于亚甲基蓝染色液结合琼脂糖法的芸薹根肿菌单孢分离方法，包括：制备芸薹根肿菌孢子悬浮液、制备亚甲基蓝琼脂糖溶液、制备亚甲基蓝琼脂糖载玻片凝层、挑取芸薹根肿菌单孢。The method for separating P. brassicae spores based on the methylene blue staining solution combined with the agarose method provided by the present invention includes: preparing a spore suspension of P. brassicae, preparing a methylene blue agarose solution, and preparing a methylene blue agarose slide glass gel Layer, pick the single spore of P. brassicae.

具体包括如下步骤：Specifically include the following steps:

1)将十字花科根肿病发病组织打成匀浆，过滤，弃沉淀，制备浓度为5×10²个孢子·mL^-1～5×10⁴个孢子·mL^-1的芸薹根肿菌孢子悬浮液，4℃～8℃保存备用，最佳保存温度为4℃；1) Homogenize the diseased tissue of cruciferous clubroot, filter, discard the precipitate, and prepare the Brassica root with a concentration of 5×10² spores·mL^-1 to 5×10⁴ spores·mL^-1 The bacterial spore suspension is stored at 4°C to 8°C for future use, and the best storage temperature is 4°C;

2)配制质量体积百分浓度为1.3％-1.5％的琼脂糖溶液，并灭菌；配制质量体积百分浓度为0.005％-0.01％的亚甲基蓝染色液；2) Prepare an agarose solution with a mass volume percent concentration of 1.3%-1.5%, and sterilize it; prepare a methylene blue staining solution with a mass volume percent concentration of 0.005%-0.01%;

将所述亚甲基蓝染色液与灭菌后的所述琼脂糖溶液按照体积比100μL：30mL的比例混合混匀，得到亚甲基蓝染色液琼脂糖溶液；Mixing the methylene blue staining solution and the sterilized agarose solution according to a volume ratio of 100 μL:30 mL to obtain a methylene blue staining solution agarose solution;

3)取洁净的载玻片，在步骤2)所述亚甲基蓝染色液琼脂糖溶液中蘸一下，使载玻片表面形成凝层；3) Take a clean glass slide and dip it in the methylene blue staining solution agarose solution described in step 2) to form a condensation layer on the surface of the slide glass;

4)将步骤1)所述的芸薹根肿菌休眠孢子悬浮液均匀涂布于亚甲基蓝染色液琼脂糖凝层表面，在显微镜下观察，确定视野下只有一个孢子时，分离含有该芸薹根肿菌单孢的亚甲基蓝染色液琼脂糖凝层，倒扣于十字花科植物的幼苗根毛处，培育得到芸薹根肿菌单孢系。4) Evenly coat the dormant spore suspension of P. brassicae described in step 1) on the surface of the agarose condensate layer of the methylene blue staining solution, observe under a microscope, and when it is determined that there is only one spore in the field of view, separate the root containing the Brassica chinensis. The agarose condensate layer of the methylene blue staining solution of P. tumefaciens is inverted on the root hairs of the seedlings of cruciferous plants, and the P. brassica monospore line is obtained by cultivation.

上述方法步骤1)与2)-3)之间无先后顺序，亦可同时进行。There is no sequence between steps 1) and 2)-3) of the above method, and can also be performed simultaneously.

上述方法步骤1)中，所述过滤的方式为采用八层纱布过滤。In step 1) of the above method, the filtering method is to filter with eight layers of gauze.

上述方法步骤2)中，所述琼脂糖溶液和亚甲基蓝染色液中的溶剂均为无菌水；所述灭菌的条件为：121℃灭菌20min。In step 2) of the above method, the solvents in the agarose solution and the methylene blue staining solution are both sterile water; the sterilization conditions are: sterilization at 121° C. for 20 minutes.

上述方法步骤2)中，将灭菌后的所述琼脂糖溶液熔化并冷至50℃-60℃，然后加入所述亚甲基蓝染色液加入所述灭菌后的琼脂糖溶液中。In step 2) of the above method, the sterilized agarose solution is melted and cooled to 50°C-60°C, and then the methylene blue staining solution is added to the sterilized agarose solution.

上述方法步骤4)中，所述的芸薹根肿菌休眠孢子悬浮液的用量为1μL～5μL。In step 4) of the above method, the dosage of the dormant spore suspension of P. brassicae is 1 μL to 5 μL.

上述方法步骤4)中，所述显微镜的最大放大倍数不低于1000倍，具体为400倍。In step 4) of the above method, the maximum magnification of the microscope is not less than 1000 times, specifically 400 times.

所述分离含有该芸薹根肿菌单孢的亚甲基蓝染色液琼脂糖凝层的方式为：用手术刀切取约1mm²含有单孢的亚甲基蓝染色液琼脂糖凝层。The method for separating the methylene blue staining solution agarose coagulation layer containing the single spore of P. brassicae is as follows: using a scalpel to cut about 1 mm² of the methylene blue staining solution agarose coagulation layer containing the single spore.

上述方法步骤4)中，所述十字花科植物具体可为菊心大白菜(购于中蔬种业科技(北京)有限公司)。所述培育的条件为：温度25℃、湿度80％、暗培养24h。In step 4) of the above-mentioned method, the cruciferous plant may specifically be Chrysanthemum Chinese cabbage (purchased from China Vegetable Seed Technology (Beijing) Co., Ltd.). The incubation conditions were: temperature 25° C., humidity 80%, and dark incubation for 24 hours.

本发明利用亚甲基蓝对琼脂糖进行染色处理后，琼脂糖凝层背景呈现轻微蓝色，而对芸薹根肿菌休眠孢子活力无影响，因此能更清晰地判断芸薹根肿菌单孢，突破性地解决了传统水琼脂法背景含杂质和气泡较多，不易区分芸薹根肿菌单孢等问题，大大提高了获取根肿菌单孢的速度及准确率，提高了获取单孢系的效率。In the present invention, after the agarose is dyed with methylene blue, the background of the agarose condensate appears slightly blue, but has no effect on the viability of the dormant spores of P. brassicae, so the single spore of P. The traditional water agar method contains many impurities and bubbles in the background, and it is difficult to distinguish the single spore of P. brassicae, greatly improving the speed and accuracy of obtaining the single spore of P. efficiency.

附图说明Description of drawings

图1为不同单孢分离方法制备的芸薹根肿菌大量孢子和单孢照片；其中A，B分别为亚甲基蓝琼脂糖法芸薹根肿菌大量孢子和单孢照片，亚甲基蓝琼脂糖背景呈轻微蓝色，且无杂质和气泡，易于区分芸薹根肿菌单孢；C，D分别为传统水琼脂法芸薹根肿菌大量孢子和单孢照片，水琼脂块背景含杂质和气泡较多，不易区分芸薹根肿菌单孢。Figure 1 is the photos of the massive spores and single spores of P. brassica prepared by different single spore separation methods; A and B are the photos of the massive spores and single spores of P. brassicae by the methylene blue agarose method, respectively. The methylene blue agarose background is slightly Blue, free of impurities and bubbles, easy to distinguish single spores of P. brassicae; C and D are photos of a large number of spores and single spores of P. brassicae by traditional water agar method, the background of water agar blocks contains more impurities and bubbles , it is not easy to distinguish single spores of P. brassicae.

具体实施方式Detailed ways

下面通过具体实施例对本发明的方法进行说明，但本发明并不局限于此，凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The method of the present invention will be described below through specific embodiments, but the present invention is not limited thereto, and any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention, etc., shall be included in the scope of the present invention. within the scope of protection.

下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

本发明提供的一种基于亚甲基蓝染色液结合琼脂糖法分离芸薹根肿菌单孢的方法，包括芸薹根肿菌的处理肿根、配制孢悬液、获取单孢子及单孢接种等过程。本发明的方法是1)将十字花科根肿病发病组织打成匀浆，八层纱布过滤，弃沉淀，制备浓度为5×10³个孢子·mL^-1的芸薹根肿菌休眠孢子悬浮液，4℃保存备用；2)取100μL的0.01％亚甲基蓝染色液加入灭菌的30mL熔化并冷至50℃左右的1.3％琼脂糖溶液内混匀。3)将灭菌的载玻片在制备好的亚甲基蓝染色液琼脂糖溶液中蘸一下，使载玻片表面形成凝层。4)然后取2μL～5μL稀释好的休眠孢子悬浮液滴于染色液琼脂糖凝层表面。在400倍显微镜下观察，确定视野只有一个孢子，用手术刀切取约1mm²含有单孢的染色液琼脂糖凝层，倒扣于感病大白菜幼苗根毛处，培育得到芸薹根肿菌单孢系。The invention provides a method for separating P. brassicae single spore based on methylene blue staining solution combined with agarose method, which includes the processes of treating P. brassicae root, preparing a spore suspension, obtaining a single spore and inoculating a single spore. . The method of the present invention is 1) making the cruciferous clubroot diseased tissue into a homogenate, filtering with eight layers of gauze, discarding the precipitation, and preparing the dormant spores of clubroot with a concentration of 5×10³ spores·mL^-1 The suspension was stored at 4°C for later use; 2) 100 μL of 0.01% methylene blue staining solution was added to 30 mL of sterilized 1.3% agarose solution melted and cooled to about 50°C and mixed. 3) Dip the sterilized glass slide in the prepared methylene blue staining solution agarose solution to form a condensation layer on the surface of the glass slide. 4) Then take 2 μL to 5 μL of the diluted dormant spore suspension drop on the surface of the staining solution agarose condensate. Observe under a 400-fold microscope to confirm that there is only one spore in the field of view. Use a scalpel to cut out about¹ mm of the agarose condensate layer of staining solution containing a single spore, and invert it at the root hair of the susceptible Chinese cabbage seedling. Spore system.

本试验以菊心大白菜(购于中蔬种业科技(北京)有限公司)为试验材料，采用传统琼脂块法和染色液琼脂糖法两种方法各接种600株大白菜幼苗，共计3次重复。接种45d左右进行调查，结果表明：采用染色液琼脂糖法挑取根肿菌单孢子不仅大大提高了接种后植株的成活率，还提高了接种后的单孢发病率，相比较发病率提高了16.71％。In this experiment, Juxin Chinese cabbage (purchased from China Vegetable Seed Industry Technology (Beijing) Co., Ltd.) was used as the test material, and 600 Chinese cabbage seedlings were inoculated each by the traditional agar block method and the dyeing solution agarose method for a total of 3 times. repeat. The investigation was carried out about 45 days after inoculation. The results showed that the single spore of P. rhizogenes was picked by the staining solution agarose method, which not only greatly improved the survival rate of the plant after inoculation, but also increased the incidence of single spore after inoculation. Compared with the increase in the incidence rate 16.71%.

实施例1、琼脂糖最佳浓度的筛选Embodiment 1, the screening of optimal concentration of agarose

将灭菌的载玻片在不同浓度琼脂糖溶液中蘸一下，使载玻片表面形成凝层。然后取5μL稀释好的休眠孢子悬浮液滴于琼脂糖凝层表面。在显微镜下观察，用手术刀切取约1mm2含有单孢的染色液的琼脂糖凝层。筛选出最佳琼脂块浓度。将琼脂糖溶液浓度设1％、1.2％、1.3％、1.5％、1.8％、2％、2.5％和3％共8个处理，分析不同浓度下琼脂糖凝层的透明度、质地硬度、以及单孢挑取的难易程度。Dip the sterilized slides in agarose solutions of different concentrations to form a coagulation layer on the surface of the slides. Then, 5 μL of the diluted dormant spore suspension was dropped on the surface of the agarose gel. Observe under a microscope, and use a scalpel to cut out about 1 mm2 of the agarose condensate layer containing the staining solution of the single spore. Screen out the best agar block concentration. The concentration of agarose solution was set to 1%, 1.2%, 1.3%, 1.5%, 1.8%, 2%, 2.5% and 3%, a total of 8 treatments were used to analyze the transparency, texture hardness, and single-layer properties of the agarose gel at different concentrations. Difficulty of picking spores.

表1不同浓度琼脂糖结果分析表Table 1 Results analysis table of different concentrations of agarose

结果显示当浓度为1.3％时，琼脂糖凝层透明度较好、质地适中、易挑取，最适合进行芸薹根肿菌单孢分离，详细结果描述见表1。The results showed that when the concentration was 1.3%, the agarose gel layer had better transparency, moderate texture and easy picking, and was the most suitable for single spore isolation of P. brassicae. The detailed results are shown in Table 1.

实施例2、最佳染色剂及染色液浓度的筛选Embodiment 2, the screening of optimal dyeing agent and dyeing solution concentration

染色液的配置：分别称取0.05g酸性品红、0.01g刚果红、0.01g伊红Y纳、0.05g中性红、0.01g番红、0.005g亚甲基蓝、0.01g亚甲基蓝、0.02g亚甲基蓝、0.03g亚甲基蓝、0.04g亚甲基蓝、0.02g溴酚蓝、0.01g甲基绿，加入100mL无菌水。配制成质量体积百分浓度分别为0.05％酸性品红、0.01％刚果红、0.01％伊红Y纳、0.05％中性红、0.01％番红、0.005％亚甲基蓝、0.01％亚甲基蓝、0.02％亚甲基蓝、0.03％亚甲基蓝、0.04％亚甲基蓝、0.02％溴酚蓝和0.01％甲基绿的染色液，置棕色瓶4℃保存备用。Configuration of staining solution: Weigh 0.05g acid fuchsin, 0.01g Congo red, 0.01g eosin Y sodium, 0.05g neutral red, 0.01g safranine, 0.005g methylene blue, 0.01g methylene blue, 0.02g methylene blue, 0.03g g methylene blue, 0.04 g methylene blue, 0.02 g bromophenol blue, 0.01 g methyl green, and 100 mL sterile water was added. The mass and volume percentage concentrations are respectively 0.05% acid fuchsin, 0.01% Congo red, 0.01% eosin Y sodium, 0.05% neutral red, 0.01% safranine, 0.005% methylene blue, 0.01% methylene blue, 0.02% methylene blue, The staining solutions of 0.03% methylene blue, 0.04% methylene blue, 0.02% bromophenol blue and 0.01% methyl green were stored in a brown bottle at 4°C for later use.

染色液琼脂糖凝层制备：取上述12种不同染色液各50μL，加入15mL熔化并冷至50℃左右的1.3％琼脂糖溶液中，置于无菌培养皿中凝固，制备染色液琼脂糖凝层。Preparation of staining solution agarose gel layer: Take 50 μL of each of the above 12 different staining solutions, add 15 mL of 1.3% agarose solution melted and cooled to about 50 °C, and place it in a sterile petri dish to solidify to prepare staining solution agarose gel. Floor.

染色液琼脂糖凝层对芸薹根肿菌孢子活力的影响：采用Hochest 33342-PI双荧光复染法，检测不同染色液处理对芸薹根肿菌休眠孢子活力的影响。将浓度1×10⁸个孢子·mL^-1的芸薹根肿菌孢子悬浮液10mL均匀涂布于各染色液琼脂糖凝层表面，对照取等量孢子悬浮液置于不加染色液的琼脂糖凝层表面，静置处理1h。取处理后的芸薹根肿菌休眠孢子悬浮液各1mL，离心弃上清，孢子沉淀先用10μg·mL^-1的Hoechst 33342染液悬浮，37℃孵育10min，再用5μg·mL^-1的PI染液4℃避光孵育15min，然后置于荧光显微镜下观察。经Hochest33342-PI双染后，有活力的休眠孢子发蓝色荧光，死的休眠孢子发红色荧光。每个处理观察记录100个休眠孢子，筛选对芸薹根肿菌休眠孢子活力无影响的染色液。结果如表2：The effect of staining solution agarose condensate on the viability of P. brassicae spores: Hochest 33342-PI double-fluorescence counterstaining method was used to detect the effects of different staining solutions on the viability of P. brassicae dormant spores. Spread 10 mL of the spore suspension of P. brassicae with a concentration of 1×10⁸ spores·mL^-1 evenly on the surface of each staining solution agarose condensate. For the control, take the same amount of spore suspension and place it on agar without staining solution. The surface of the sugar condensate layer was left to stand for 1 h. Take 1 mL of the treated suspension of dormant spores of P. brassicae, centrifuge to discard the supernatant, and suspend the spore pellet with 10 μg·mL^-1 of Hoechst 33342 staining solution, incubate at 37°C for 10 min, and then use 5 μg·mL^-1 of The PI staining solution was incubated at 4°C for 15 min in the dark, and then placed under a fluorescence microscope for observation. After double staining with Hochest33342-PI, viable dormant spores fluoresced blue, and dead dormant spores fluoresced red. 100 dormant spores were observed and recorded in each treatment, and the staining solution that had no effect on the viability of the dormant spores of P. brassicae was screened. The results are shown in Table 2:

表2不同染色剂对芸薹根肿菌休眠孢子活力影响Table 2 Effects of different dyes on the viability of dormant spores of P. brassicae

表3不同浓度亚甲基蓝染色液对芸薹根肿菌休眠孢子活力影响Table 3 Effects of different concentrations of methylene blue staining solution on the viability of dormant spores of P. brassicae

如表2、表3所示：不同染色液对芸薹根肿菌休眠孢子活力有一定的影响。其中亚甲基蓝染色液对根肿菌休眠孢子活力影响最小，以无菌水为溶剂的0.01％亚甲基蓝染色液处理休眠孢子存活率为97％，以PBS缓冲液为溶剂的0.01％亚甲基蓝染色液处理休眠孢子存活率为95％，与对照不加染色剂的处理孢子存活率98％差异不显著。在一定范围内，随着以无菌水为溶剂亚甲基蓝染色液浓度的增加，经处理后休眠孢子的成活率下降。酸性品红、溴酚蓝、甲基绿等，对芸薹根肿菌孢子活力都有一定的影响，休眠孢子存活率在61％～87％之间。此外，将芸薹根肿菌置于亚甲基蓝染色液琼脂糖凝层处理后，在光学显微镜下观察，琼脂糖背景和芸薹肿根残留组织菌被染为淡蓝色，而有活力的根肿菌孢子不被染色，因此便于区分芸薹根肿菌单孢。综上所述，亚甲基蓝染色液对芸薹根肿菌孢子活力无影响，可用于单孢分离是琼脂糖凝层背景的染色(见图1)。As shown in Table 2 and Table 3: different staining solutions have a certain influence on the viability of dormant spores of P. brassicae. Among them, the methylene blue staining solution had the least effect on the viability of the dormant spores of P. rhizogenes. The survival rate of the dormant spores was 97% when treated with the 0.01% methylene blue staining solution with sterile water as the solvent, and the dormant spores were treated with the 0.01% methylene blue staining solution with PBS buffer as the solvent. The survival rate was 95%, which was not significantly different from the 98% survival rate of spores treated with no staining agent. Within a certain range, the survival rate of dormant spores decreased after treatment with the increase of the concentration of methylene blue staining solution using sterile water as solvent. Acid fuchsin, bromophenol blue, methyl green, etc., all had certain effects on the spore viability of P. brassicae, and the survival rate of dormant spores was between 61% and 87%. In addition, after placing Plasmoradicula Brassica in the agarose condensate layer of methylene blue staining solution, observed under a light microscope, the agarose background and the residual tissue bacteria of the Brassica rhizogenes were stained light blue, while the viable tumor roots were stained light blue. The spores are not stained, thus facilitating the identification of P. brassica monospores. In conclusion, the methylene blue staining solution has no effect on the spore viability of P. brassicae, and can be used for single spore isolation and staining against the background of agarose gel (see Figure 1).

实施例3、亚甲基蓝琼脂糖块法与传统水琼脂法单孢分离效果的比较Example 3. Comparison of single spore separation effect between methylene blue agarose block method and traditional water agar method

采用本发明建立的亚甲基蓝琼脂糖块法与已报道的水琼脂法(背景技术中提及的方法一)进行芸薹根肿菌单孢接菌，比较根肿菌单孢分离结果。对催芽24h的菊心大白菜(购于中蔬种业科技(北京)有限公司)幼苗利用2种不同的方法接种处理后，移植到直径5cm×5cm无菌育苗盘，置于温室中培养。各处理200株，3次重复。置于温室环境中，保持日温20～25℃、夜温11～16℃、光照16h·d^-1，前两周湿度100％，之后按需浇水，温室统一管理。The methylene blue agarose block method established by the present invention and the reported water agar method (method 1 mentioned in the background art) were used to inoculate P. brassicae monospores, and the isolation results of P. rhizogenes monospores were compared. Chrysanthemum Chinese cabbage seedlings (purchased from China Vegetable Seed Industry Technology (Beijing) Co., Ltd.) that had been primed for 24 hours were inoculated by two different methods, and then transplanted into sterile seedling trays with a diameter of 5cm×5cm and cultured in a greenhouse. 200 plants were treated with 3 replicates each. Placed in a greenhouse environment with a day temperature of 20-25°C, night temperature of 11-16°C, light of 16h·d^-1 , 100% humidity for the first two weeks, and then watered as needed, and the greenhouse was managed uniformly.

表4水琼脂块法接种与亚甲基蓝琼脂糖块法接种调查数据统计Table 4 Survey data statistics of water agar block method inoculation and methylene blue agarose block method inoculation

结果如表4所示：同等栽培条件下，采用相同的菌株和接种寄主材料，比较传统水琼脂块法和亚甲基蓝琼脂糖块法接种单孢后大白菜的发病情况，即获得芸薹根肿菌单孢株系情况。通过试验结果对比，传统琼脂糖法单孢发病率为0.74％，共获得单孢株系3株；而本发明所用的染色液琼脂糖块法的发病率为17.45％，共获得单孢株系85株，远远高于传统水琼脂块法的寄主发病率和单孢株系获得概率。结果表明本发明基于亚甲基蓝琼脂糖块法的芸薹根肿菌单孢分离方法，大大提高了接种寄主大白菜的发病率和芸薹根肿菌单孢获得概率，该方法是可靠、可行的。The results are shown in Table 4: Under the same cultivation conditions, the same strains and inoculated host materials were used to compare the incidence of Chinese cabbage after inoculation with single spores by the traditional water agar block method and the methylene blue agarose block method. Single spore strains. By comparing the test results, the traditional agarose method has a single spore incidence rate of 0.74%, and a total of 3 single spore strains have been obtained; while the staining solution agarose block method used in the present invention has an incidence rate of 17.45%, and a total of single spore strains have been obtained. 85 strains, which is much higher than the traditional water agar block method for the incidence of host and the probability of obtaining a single spore strain. The results show that the method for isolating P. brassica monospores based on the methylene blue agarose block method of the present invention greatly improves the incidence of inoculated host Chinese cabbage and the probability of obtaining P. brassica monospores, and the method is reliable and feasible.

采用本发明的方法适于挑取芸薹根肿菌单孢子，用琼脂糖代替水琼脂，无杂质；加入染色液后，能更块、更准确地观察到单孢；且染色液对孢子活力无影响。可以从根本上解决不易分离芸薹根肿菌单孢的操作问题及接种后大白菜不易成活的问题。The method of the invention is suitable for picking single spores of P. brassicae, and agarose is used instead of water agar, without impurities; after adding the dyeing solution, the single spore can be observed in more pieces and more accurately; and the dyeing solution has a positive effect on the spore vitality no effect. It can fundamentally solve the operation problem that it is difficult to separate the single spore of P. brassicae and the problem that the Chinese cabbage is not easy to survive after inoculation.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何不经过创造性活动想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求书所限定的保护范围为准。The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto, and any changes or substitutions that are not conceived through creative activities should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined by the claims.

Claims (10)

1. A plasmodiophora brassicae monospore separation method based on a methylene blue staining solution combined agarose method comprises the following steps:

1) beating the cruciferous clubroot disease-affected tissues into homogenate, filtering, and discarding the precipitate to prepare a plasmodiophora brassicae spore suspension;

2) preparing an agarose solution with the mass volume percentage concentration of 1.3-1.5%, and sterilizing;

preparing methylene blue staining solution with the mass volume percentage concentration of 0.005-0.01%;

and (3) mixing the methylene blue staining solution with the sterilized agarose solution according to the volume ratio of 100 mu L: mixing and uniformly mixing the materials in a proportion of 30mL to obtain a methylene blue staining solution agarose solution;

3) dipping a clean glass slide in the methylene blue staining solution agarose solution obtained in the step 2) to form a gel layer on the surface of the glass slide;

4) uniformly coating the plasmodiophora brassicae resting spore suspension obtained in the step 1) on the surface of a methylene blue staining solution agarose gel layer, observing under a microscope, separating the methylene blue staining solution agarose gel layer containing plasmodiophora brassicae monospore when only one spore is determined in a visual field, inversely covering the agarose gel layer on the root hair of seedlings of cruciferous plants, and culturing to obtain the plasmodiophora brassicae monospore system.

2. The separation method according to claim 1, characterized in that: in the step 1), the filtering mode is filtering by adopting eight layers of gauze.

3. The separation method according to claim 1 or 2, wherein in the step 1), the concentration of spores in the plasmodiophora brassicae resting spore suspension is 5 × 10²Spore. mL^-1～5×10⁴Spore. mL^-1。

4. The separation method according to claim 1, characterized in that: in the step 1), the plasmodiophora brassicae spore suspension is stored at 4-8 ℃ before use.

5. The separation method according to claim 1, characterized in that: in the step 2), solvents in the agarose solution and the methylene blue staining solution are sterile water; the sterilization conditions are as follows: sterilizing at 121 deg.C for 20 min.

6. The separation method according to claim 1, characterized in that: in the step 2), the sterilized agarose solution is melted and cooled to 50-60 ℃, and then the methylene blue staining solution is added.

7. The separation method according to claim 1, characterized in that: in the step 4), the magnification of the microscope is 400 times.

8. The separation method according to claim 1, characterized in that: in the step 4), the manner of separating the methylene blue staining solution agarose gel layer containing the plasmodiophora brassicae monospore is as follows: cutting about 1mm with a scalpel²And (3) carrying out agarose gel layer by methylene blue staining solution containing monospore.

9. The separation method according to claim 1, characterized in that: in the step 4), the cruciferous plants are Chinese cabbages.

10. The separation method according to claim 9, characterized in that: in the step 4), the cultivation conditions are as follows: culturing at 25 deg.C and 80% humidity in dark for 24 hr.

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