Convenient method for screening facultative anaerobic protozoa in rice field soil

Abstract

The invention provides a convenient method for screening facultative anaerobic protozoa in rice field soil, and the method effectively creates an environment suitable for growth and reproduction of facultative anaerobic protozoa by collecting and processing a rice field soil sample and utilizing a simple anaerobic culture device. Compared with a traditional screening method, the method has the advantages that the experimental operation process is simplified, complicated and expensive instruments and equipment are not needed, and the experimental cost and the technical difficulty are greatly reduced. And meanwhile, the screening efficiency is remarkably improved, and the facultative anaerobic protozoa sample with higher purity can be obtained in a shorter time.

Description

Technical Field

[0001] The invention relates to the technical field of agriculture and is a convenient method for screening facultative anaerobic protozoa in paddy field soil. Background Art

[0002] In the soil ecosystem, which is highly complex and diverse in species, soil protozoa are tiny in size, but they play an irreplaceable and key role in material circulation and energy conversion. In terms of material circulation, they participate in key processes such as carbon cycle, nitrogen cycle and phosphorus cycle to promote carbon turnover rate, nitrogen utilization rate of plants and phosphorus activation efficiency. In terms of energy conversion, soil protozoa successfully release the chemical energy stored in organic matter by oxidizing and decomposing organic matter. In this process, part of the energy is used to meet the needs of their own metabolism, growth and reproduction and other life activities to ensure the continuation and development of their own population; while the other part of the energy is dissipated into the soil environment in the form of heat energy, etc., providing necessary energy support for various biochemical processes in the soil ecosystem. In addition, soil protozoa also play an indispensable role in maintaining soil fertility, gradually decomposing complex organic matter into simple inorganic matter, releasing nitrogen, phosphorus, potassium and other nutrients necessary for plant growth, and providing sufficient nutrition sources for the healthy growth of plants. Not only that, soil protozoa also have the special ability to synthesize biologically active substances, such as plant hormones, vitamins and other substances that play an important role in promoting plant growth and development.

[0003] As a type of unicellular eukaryotic organisms, the cell structure and metabolic system of facultative anaerobic protozoa have been highly adapted to anaerobic environments during the long-term evolution process, enabling them to exhibit unique material metabolism capabilities under anaerobic conditions. From the perspective of cellular metabolic mechanisms, they can utilize complex organic matter in the soil and convert it into simpler compounds through a series of special metabolic pathways, while obtaining the energy required for their own growth and reproduction in the process. In terms of ecological processes, there are complex interactions between anaerobic protozoa and other microorganisms. For example, they may form a symbiotic relationship with anaerobic bacteria and cooperate with each other to complete the metabolism and transformation of certain substances; or they may regulate the structure and function of soil microbial communities and maintain the balance of the ecosystem by preying on other microorganisms. Therefore, in-depth research on the richness, diversity and ecological functions of facultative anaerobic protozoa in the soil is of great significance for our comprehensive and in-depth understanding of the operating mechanism of the soil ecosystem and the principle of maintaining ecological balance.

[0004] However, in current research and practical applications, the screening of facultative anaerobic protozoa in paddy soil faces many difficulties. Traditional screening techniques are highly dependent on various complex instruments and equipment, such as specialized anaerobic incubators, high-precision microscopic imaging systems, and cell separation devices that require precise control. These devices not only have high purchase and operating costs, but also require regular maintenance and replacement of parts, which undoubtedly greatly restricts some laboratories or research institutions with limited resources from conducting related research. At the same time, the operation process of traditional screening methods is cumbersome and complex, and has extremely high requirements on the professional quality and experimental skills of researchers. From the collection, pretreatment, anaerobic culture of paddy soil samples, to the separation and identification of facultative anaerobic protozoa, researchers need to strictly control the culture conditions and accurately adjust parameters such as temperature, humidity, and gas composition. Any slight deviation may lead to changes in the growth environment of anaerobic protozoa, thereby affecting the experimental results. In addition, the screening efficiency of traditional methods is generally low, and it often takes a lot of time and effort to obtain a small amount of anaerobic protozoa samples. Moreover, the samples obtained are often mixed with other protozoa, and the purity cannot reach the ideal level. This has brought great trouble to subsequent in-depth research and practical applications, and it is difficult to meet the urgent needs of today's scientific research and practical applications for high-quality anaerobic protozoa samples.

[0005] In summary, it is urgent to explore and develop a screening method for facultative anaerobic protozoa in paddy field soil that is portable, efficient and low-cost. If this innovative screening method can be successfully developed and vigorously promoted, it will undoubtedly bring the research in the field of facultative anaerobic protozoa in soil to a climax. With the help of this efficient and convenient screening method, researchers can obtain high-quality facultative anaerobic protozoa samples more quickly and accurately, greatly shorten the research cycle, reduce research costs, and accelerate the in-depth exploration of the types, quantity, ecological functions and metabolic mechanisms of soil protozoa. This will not only help us understand the operating laws of soil ecosystems more comprehensively and deeply, but also provide key technical support for solving a series of scientific problems related to soil ecology. Summary of the invention

[0006] The present invention solves the technical difficulties currently faced in screening facultative anaerobic protozoa in paddy soil, namely, the traditional screening technology relies on expensive instruments and equipment, has high purchase and maintenance costs, has cumbersome and complicated operation procedures, requires extremely high professional skills of researchers, and has low screening efficiency, and discloses a convenient screening method. The method adopts a step-by-step transition method, which can achieve efficient screening of anaerobic protozoa in paddy soil, is not only easy to operate, but also greatly reduces the screening cost.

[0007] A convenient method for screening facultative anaerobic protozoa in paddy soil, the technical scheme adopted is: A convenient method for screening facultative anaerobic protozoa in paddy field soil aims to provide an innovative and easy-to-operate method for screening facultative anaerobic protozoa in paddy field soil, effectively solving the problems of complex operation, high cost, low efficiency and low sample purity existing in the prior art.

[0008] This technical solution can effectively screen facultative anaerobic protozoa in paddy field soil.

[0009] Secondly, it is difficult to create an anaerobic environment. It is often necessary to use anaerobic incubators and anaerobic gas production bags to ensure an anaerobic environment and prevent oxygen from entering and affecting the reproduction of anaerobic protozoa. This method requires high equipment. This method does not need to consider equipment issues. It can be achieved with simple equipment, is easy to operate, and will not affect the survival of protozoa.

[0010] In addition, the screening method specifically targeting facultative anaerobic protozoa can effectively separate facultative anaerobic protozoa from paddy field soil samples, avoiding interference from other non-target organisms such as aerobic organisms, and improving the accuracy and reliability of screening.

[0011] Therefore, this method is more conducive to the screening and reproduction of facultative anaerobic protozoa.

[0012] As a further improvement of the present invention, the protozoa are firstly cultured in vivo by non-submerged culture method to obtain living culture fluid, then transferred to a micro-aerobic seed bottle for culture, and finally transferred to an anaerobic culture bottle for mass reproduction.

[0013] As a further improvement of the present invention, the facultative anaerobic protozoa feed on rice grain extract and are cultured in large quantities in a constant temperature incubator at 20°C.

[0014] As a further improvement of the present invention, the number of the protozoa reaches a peak after about one week of cultivation, then decreases and approaches disappearance, and recovers and maintains stability after one month.

[0015] Compared with the prior art, the present invention has the following beneficial effects: By adopting the technical solution of the present invention, the operation process of the method is simplified, and no complicated and expensive instruments and equipment are required, which greatly reduces the experimental cost and technical difficulty. At the same time, the screening efficiency is significantly improved, and a facultative anaerobic protozoan sample with high purity can be obtained in a relatively short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a diagram of an implementation scheme for micro-oxygen experiment according to an embodiment of the present invention.

[0017] Figure 2 It is a diagram of an implementation scheme for anaerobic experiment according to an embodiment of the present invention.

[0018] Figure 3 It is a living picture of protozoa screened out in the embodiments of the present invention.

[0019] Figure 4 It is a silver staining picture of the protozoan protein screened in the embodiment of the present invention.

[0020] Figure 5 It is the evolutionary tree of protozoa appearing in the embodiments of the present invention. DETAILED DESCRIPTION

[0021] The preferred embodiments of the present invention are described in further detail below.

[0022] The soil sample used in the following examples is rice rhizosphere soil.

[0023] Specifically include: 1. Preparation of soil leachate Take 30 g of paddy field soil sample, place it in a beaker, add 100 mL of distilled water, boil for 10 min to 15 min, let it stand to settle, and filter the suspension to obtain the soil extract.

[0024] 2. Non-submerged culture method Take another 5 g paddy field soil sample, wrap it with gauze and put it in a culture dish, add about 60 mL of soil extract, and the extract should cover 2 / 3 of the sample. Culture the protozoa in the paddy field soil and culture it in a constant temperature incubator at 20 ℃ for 3 days to obtain the living culture fluid.

[0025] 3. Gradually transition to screening anaerobic protozoa Transfer the living culture medium to a micro-aerobic seed bottle and culture for at least one month. Figure 1 shown.

[0026] Transfer the culture medium in the micro-aerobic seed bottle to an anaerobic culture bottle filled with 400 cc N2 and feed it with rice grain extract. Figure 2 shown.

[0027] After one week, measure the oxygen concentration and check for the presence of protozoa. The existing protozoa are transferred to anaerobic culture bottles filled with 400 cc N2 and fed with rice grain extract for monoclonal culture. After obtaining high-purity anaerobic protozoa, morphological identification is performed. Figure 3 , Figure 4 shown.

[0028] Species Identification The selected facultative anaerobic protozoa were identified by molecular biology. Figure 5 shown.

[0029] Experimental results: Screening out a facultative anaerobic protozoa.

[0030] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A convenient method for screening facultative anaerobic protozoa in paddy soil, characterized in that: Through the collection and processing steps of paddy field soil samples and the use of a simple anaerobic culture device, an environment suitable for the growth and reproduction of facultative anaerobic protozoa was effectively created. Specifically, it includes: (1) Preparation of soil leachate; (2) Non-submerged culture method; (3) Gradually transition to screening for anaerobic protozoa; (4) Species identification.

2. The convenient method for screening facultative anaerobic protozoa in paddy field soil according to claim 1, characterized in that: The facultative anaerobic protozoa feed on rice grain extract and are cultured in large quantities in a constant temperature incubator at 20°C.

3. The convenient method for screening facultative anaerobic protozoa in paddy field soil according to claim 2, characterized in that: After reaching a peak in about a week of cultivation, the population decreased and almost disappeared. After a month, the population recovered and remained stable.

4. The convenient method for screening facultative anaerobic protozoa in paddy field soil according to claim 1, characterized in that: The protozoa is firstly cultured by a non-submerged living culture method to obtain a living culture fluid, then transferred to a micro-aerobic seed preservation bottle for culture, and finally transferred to an anaerobic culture bottle for mass reproduction.

5. Screen out the use of facultative anaerobic protozoa, characterized in that: The protozoa can be applied to soil ecological restoration, improving soil structure and fertility by participating in material circulation and energy conversion; because of their high sensitivity and strong indicative characteristics to pollutants, they can be used as indicator biological reactions to soil pollution levels and ecological health conditions and can be applied to the field of environmental monitoring.

6. The use of facultative anaerobic protozoa according to claim 5, characterized in that: The facultative anaerobic protozoa feed on rice grain extract and are cultured in a constant temperature incubator at 20° C. The experimental operation is simple, does not require complex and expensive equipment and technology, and has low cost.

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