A method for rapid field extraction of dna from plant bacterial disease lesions

Abstract

The application belongs to the technical field of DNA extraction, and particularly relates to a field rapid extraction method of DNA in plant bacterial disease lesion, which comprises the following steps: mixing plant disease leaf lesion tissue and a lysis solution, and heating in a water bath in a thermos cup to complete nucleic acid release; the lysis solution comprises 0.05-0.2 mol / L NaOH; preferably, the lysis solution comprises 0.05-0.2 mol / L NaOH, 0.01% SDS and 0.1 mmol / L EDTA. The DNA extraction method established by the application does not need precise instruments and toxic reagents, is simple to operate, short in time consumption, and can realize field rapid extraction of plant lesion bacterial DNA, thereby providing technical support for early diagnosis and accurate prevention and control of plant bacterial diseases in the field.

Description

Technical Field

[0001] This invention belongs to the field of DNA extraction technology, and more specifically, relates to a rapid field extraction method for DNA from lesions of bacterial plant diseases. Background Technology

[0002] Bacterial diseases are a significant threat to global agricultural production. They are widespread, spread rapidly, and have diverse transmission routes, often leading to sharp declines in crop yields and quality. Of the known plant infectious diseases, approximately 7% are caused by bacteria, and these bacterial diseases typically result in yield reductions of 10% to 30%, with losses exceeding 50% or even total crop failure in severe cases, seriously hindering the improvement of agricultural production efficiency. In recent years, with global warming and changes in intensive farming practices, various bacterial diseases, such as bacterial angular leaf spot of cucumber and bacterial spot of soybean, have shown an increasing trend in major crop-producing areas. In some areas, the severity of these diseases can reach 50%, with yield losses of 30% to 50%.

[0003] Traditional methods for identifying plant pathogenic bacteria mainly include pathogen isolation and culture, physiological and biochemical assays, and pathogenicity determination. These methods rely on the in vitro culture characteristics of pathogens, which are not only time-consuming (usually requiring 3-7 days, and even more than 10 days for complex samples), but also cumbersome, requiring multiple steps such as isolation and purification, biochemical index detection, and Koch's postulate verification. They are highly dependent on specialized laboratory equipment and experienced technicians. In field disease outbreaks, the lag of traditional methods can easily lead to delays in control measures, resulting in the spread of the disease and failing to meet the urgent need for rapid field diagnosis. With the development of molecular biology techniques, polymerase chain reaction (PCR)-based molecular detection technology has become the mainstream method for identifying plant pathogens due to its high sensitivity, strong specificity, and short detection cycle, providing technical support for early disease diagnosis. However, PCR detection requires obtaining a high-quality DNA template from the sample, and currently widely used DNA extraction methods all have significant limitations. Whether it's the classic CTAB method, SDS method, or commercially available reagent kits, all methods rely on sophisticated laboratory equipment such as centrifuges, water baths, and vortex mixers. They also involve complex steps like phenol-chloroform extraction, ethanol precipitation, and centrifugal purification, resulting in cumbersome and time-consuming procedures. Furthermore, organic solvents like phenol and chloroform are toxic, posing safety risks. These limitations hinder the widespread application of existing methods in field settings, grassroots plant protection stations, and resource-scarce areas, becoming a major obstacle to the practical implementation of molecular diagnostic technologies for plant bacterial diseases.

[0004] Plant diseased tissues, especially lesions, have a complex and unique composition. Besides the nucleic acid of the target pathogen, they contain a large amount of plant polysaccharides, polyphenols, proteins, tannins, flavonoid secondary metabolites, and various PCR inhibitors. These substances not only bind to DNA to form complexes, affecting DNA release and purification, but also inhibit the activity of polymerases in the PCR reaction, severely interfering with the efficiency and accuracy of subsequent molecular detection, leading to false negative or false positive results. Therefore, how to rapidly and easily extract bacterial DNA of sufficient purity suitable for PCR amplification from plant lesions under non-laboratory conditions, without complex instruments and toxic reagents, has become an urgent problem to be solved in the development of field molecular diagnostic technology for plant diseases. Summary of the Invention

[0005] This invention addresses the problems of reliance on sophisticated instruments, cumbersome procedures, and susceptibility to interference from impurities in the field molecular diagnosis of plant bacterial diseases. It establishes a direct and rapid method for extracting bacteria directly from plant lesions, suitable for field environments. This DNA extraction method requires no sophisticated instruments or toxic reagents, is simple to operate, and is quick, enabling rapid on-site extraction of bacterial DNA from plant lesions. This provides technical support for early field diagnosis and precise control of plant bacterial diseases.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for rapid field extraction of DNA from lesions of bacterial plant diseases, comprising the following steps: mixing diseased leaf lesion tissue with lysis buffer, heating in a water bath in an insulated cup to release nucleic acids; wherein the lysis buffer comprises 0.05~0.2 mol / L NaOH.

[0007] In a preferred embodiment, the lysis buffer comprises 0.05~0.2 mol / L NaOH, 0.01% SDS, and 0.1 mmol / L EDTA.

[0008] In the preferred embodiment, the plant species are cucumber, sesame, green bean, soybean-E, rice, sorghum, soybean-G, or soybean-D.

[0009] Under the preferred scheme, the bacterial disease affecting plants is *Pseudomonas syringae* (…). Pseudomonas syringae Pantotheca ( ) Pantoea sp. ) or Xanthomonas oryzae ( Xanthomonas campestris ).

[0010] In the preferred embodiment, the water bath heating conditions inside the thermos are 95~100℃ for 10~15 minutes.

[0011] A second aspect of the present invention provides a method for rapid detection of DNA in lesions of plant bacterial diseases, characterized by comprising the following steps: S1. Nucleic acid release is completed using any of the methods described above to obtain an extract; S2. Dilute the extract 5 to 10 times; S3. Use the diluted extract as a DNA template for PCR detection.

[0012] Beneficial effects of the present invention This invention provides a lysis buffer suitable for extracting bacterial DNA from plant lesions in a field environment. The lysis buffer contains 0.05~0.2 mol / L NaOH. This lysis buffer can successfully extract bacterial DNA templates suitable for PCR amplification from diseased leaf lesion tissue. Further optimization of the composition of the lysis buffer by adding 0.01% SDS and 0.1 mmol / L EDTA significantly improves the concentration of DNA obtained from plant lesion tissue, making it suitable for detecting pathogenic bacteria in diseased leaves of plants with low bacterial loads.

[0013] This invention uses an insulated cup instead of a traditional water bath and simplifies the sample processing procedure, enabling bacterial DNA preparation that is simple to operate, time-saving, and low-cost, without the need for sophisticated instruments. This method is expected to efficiently and stably obtain qualified DNA templates in field settings or where resources are limited, providing technical support for the on-site molecular detection of plant bacterial diseases. It also provides methodological reserves for the improvement and construction of grassroots disease prevention and control systems, contributing to the green and sustainable development of agriculture.

[0014] This invention focuses on bacterial angular leaf spot of cucumber and establishes a rapid method for extracting bacterial DNA from plant lesions in the field. The method was validated using samples of different bacterial diseases from various regions across the country, demonstrating its versatility and ability to effectively extract bacterial disease DNA from lesions of crops such as cucumber, soybean, and rice. The method is also compatible with PCR detection and strain molecular identification. Attached Figure Description

[0015] Figure 1 The results of PCR detection of DNA extracted from cucumber leaves in Example 1 are shown in Figure 1. A: M is a 2000bp DNA Marker, 1 is a positive strain DNA control, 2 is bacterial DNA extracted from cucumber lesion tissue using lysis buffer L1, 3 is DNA extracted from healthy cucumber leaf tissue, and CK is a ddH2O negative control. B is a picture of the thermos cup device. Figure 2 Example 1: Symptoms of bacterial angular leaf spot in cucumber and morphological diagram of the pathogen; A: Phenotypic diagram of diseased leaves of bacterial leaf spot in cucumber; B: Strand pattern of pathogen isolated on plate; C: Gram staining morphology diagram of pathogen. Figure 3 Based on the pathogen of bacterial angular leaf spot in cucumber 16S rRNAPhylogenetic tree of genes; Figure 4 Phenotypic diagrams of diseased plants from 8 plant species; AH represent cucumber, sesame, green bean, soybean-E, rice, sorghum, soybean-G, and soybean-D, respectively. Figure 5 The results of PCR detection of DNA extracted from leaves of different plants in Example 2 are shown in A: M is a 2000bp DNA Marker, 1 is the DNA control of positive strains, 2-9 and 10-17 are cucumber, sesame, green bean, soybean-E, rice, sorghum, soybean-G, and soybean-D, respectively; 2-9 are lesion tissues, and 10-17 are healthy leaf tissues. Figure 6 Based on various different plant bacterial pathogens 16S rRNA Phylogenetic tree of genes; HRHG2: cucumber, ZM1: sesame, CD1: green bean, DD2: soybean-E, SD1: rice, GL1: sorghum, DD3: soybean-G, DD1: soybean-D; Figure 7 The images show the PCR amplification results of DNA extracted from cucumber leaves using lysis buffers with six different components; AC: M is a 2000bp DNA marker, 1 is the DNA control from a positive strain, 2-7 are bacterial DNA extracted from cucumber lesion tissue using lysis buffers L2, L3, L4, L5, L6, and L7 respectively, 8-13 are DNA extracted from healthy cucumber leaf tissue using lysis buffers L2, L3, L4, L5, L6, and L7 respectively, and CK is the ddH2O negative control; A is the electrophoresis image of PCR amplification based on primers 27F / 1492R; B is the electrophoresis image of PCR amplification based on primers Ps-F / Ps-R; C is the electrophoresis image of PCR amplification based on primers rpoD-F / rpoD-R. Figure 8 The results show the PCR amplification of DNA extracted from cucumber leaves before and after dilution using lysis buffers with seven different NaOH concentrations; AE:M represents a 2000bp DNA marker, 1 is the DNA control from a positive strain, 2-8 are bacterial DNA extracted from cucumber lesion tissue using lysis buffers L8, L9, L2, L10, L11, L12, and L13 respectively, 9-15 are DNA extracted from healthy cucumber leaves using lysis buffers L8, L9, L2, L10, L11, L12, and L13 respectively, CK is the ddH2O negative control; A is undiluted, B is diluted 1-fold, C is diluted 3-fold, D is diluted 5-fold, and E is diluted 10-fold. Figure 9PCR amplification results of DNA extracted from cucumber leaves using different lysis buffers after optimization of lysis buffer composition and DNA extraction process; AC: M is a 2000bp DNA Marker, 1 is a positive strain DNA control, 2-4 are bacterial DNA extracted from cucumber lesion tissue using lysis buffers L9, L2, and L10 respectively, 5-7 are DNA extracted from healthy cucumber leaves using lysis buffers L9, L2, and L10 respectively, CK is a ddH2O negative control; DF: M is a 2000bp DNA Marker, 1 is a positive strain DNA control, 2-4 are bacterial DNA extracted from cucumber lesion tissue using lysis buffers L1, L14, and L15 respectively, 5-7 are DNA extracted from healthy cucumber leaves using lysis buffers L1, L14, and L15 respectively, CK is a ddH2O negative control; A and D are undiluted, B and E are 5-fold diluted, and C and F are 10-fold diluted. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0017] Test materials The cucumber leaves infected with bacterial angular leaf spot used in this invention were collected from the planting field in Daqing City, Heilongjiang Province; the bacterial genome extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the PCR primers were synthesized by Qingke Biotechnology; sterile quartz sand, NaOH, NaCl, KCl, mannitol, glutamic acid, cysteine, glycine, ascorbic acid, phosphate buffer, bovine serum albumin, Tween-20, polyvinyl pyrrolidone 40 (PVP 40), trimethylol aminomethane (Tris-base), cetyltrimethylammonium bromide (CTAB), ethylenediaminetetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0018] Example 1

[0019] 1. Reagent and Sample Preparation 1) Reagents Lysis buffer L1: 0.05 mol / L NaOH, 0.01% SDS, 0.1 mmol / L EDTA; after filtration through a 0.22 μm filter membrane and sterilization, the prepared lysis buffer was dispensed into centrifuge tubes, 300 μL per tube, the tubes were tightly capped and stored at room temperature for later use.

[0020] 2) Sample Lesion tissue: Take cucumber leaves with bacterial angular leaf spot, rinse with running water to remove particles adhering to the surface of the diseased leaves, then remove the surface moisture, and use a punch to take lesion tissue with a diameter of 6 mm from the symptom area of ​​the diseased leaf to obtain cucumber diseased leaf lesion tissue.

[0021] Healthy tissue: Take healthy cucumber leaves and process them according to the treatment method for diseased tissue described above to obtain healthy cucumber leaf tissue.

[0022] 2. Bacterial DNA extraction Diseased cucumber leaf lesion tissue was added to a centrifuge tube containing lysis buffer. The centrifuge tube was placed in a thermos and heated at 95°C for 10 minutes to lyse the lesions, thus releasing the nucleic acid and obtaining the extract. Healthy cucumber leaf tissue was treated in the same way.

[0023] 3. PCR detection To evaluate the effect of the lysis buffer on the release of bacterial DNA from diseased leaf spot tissue in cucumbers, the obtained extract was diluted 5 times. Using the diluted extract as a template, bacteria were then... 16S rRNA PCR detection was performed using universal primers 27F / 1492R. The primer sequences, reaction system, and procedure used for PCR detection are as follows: Table 1. Nucleotide sequences of universal bacterial primers

[0024] Table 2 PCR detection reaction system

[0025] Program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles, and finally 72℃ extension for 8 min.

[0026] The amplification products were detected by 2% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown. By Figure 1 As can be seen, bacterial DNA extracted using L1 as the lysis buffer, after being diluted 5-fold and subjected to PCR amplification, yielded a clear target band, indicating that lysis buffer L1 can effectively extract pathogenic bacterial DNA from diseased leaf lesions in cucumbers. Furthermore, the method used did not employ any specialized laboratory equipment, eliminating reliance on traditional equipment. The heating process used an insulated cup instead of a traditional water bath, providing the necessary temperature and environment for DNA extraction. The operation is simple, rapid, and more suitable for extracting pathogenic bacterial DNA from diseased leaves in field conditions.

[0027] Preliminary detection of bacterial DNA in cucumber leaf spots was performed using gel electrophoresis. 16S rRNAThe size and purity of the target band obtained by gene PCR amplification were determined, and the sequence was then sequenced. The sequenced sequences were entered into the NCBI website for BLAST homology comparison to obtain similarity. Based on the results, a phylogenetic tree was constructed, and the results are as follows: Figure 6 The comparison results of HRHG2 identified the extracted plant bacterial pathogen as *Pseudomonas syringae*. Pseudomonas syringae ).

[0028] 4. Verification of bacterial plant diseases The above-mentioned identification results of this embodiment were verified using conventional methods, and the process is as follows: Cucumber plants exhibiting natural disease and typical leaf spot symptoms were selected, placed in sterile sampling bags, stored at low temperature, and brought back to the laboratory for processing as soon as possible. Small pieces of diseased tissue were taken, surface-sterilized, and washed twice with sterile water. The tissue was then crushed in sterile water on a sterile glass slide using a sterile glass rod. After standing for a certain period, 100 µL of the tissue fluid was spread onto LB agar and incubated at 27°C for 24 hours. Single colonies were selected based on colony morphology and color for purification culture.

[0029] Morphological characteristics of the strain were determined according to methods described in the literature. Molecular identification of the strain involved the extraction of genomic DNA using a bacterial genome extraction kit. 16S rRNA The gene was amplified by PCR, and the size and purity of the target band were initially detected by gel electrophoresis. Sequencing was then performed, and the sequenced sequences were entered into the NCBI website for BLAST homology comparison to obtain similarity. Based on the results, a phylogenetic tree was constructed.

[0030] Symptoms of cucumber bacterial angular leaf spot include: Figure 2 As shown in Figure A, the leaves exhibit typical polygonal yellowish-brown lesions with yellow halos around the edges. After surface disinfection, tissue grinding, and purification on LB medium, the pathogenic strain was obtained, as follows: Figure 2 As shown in Figure B, the pathogenic bacterial strain forms milky-white, round, raised colonies with neat edges on LB nutrient agar plates; Gram staining and observation of bacterial morphology reveal that the bacteria are rod-shaped with blunt, rounded ends, and the staining result is negative. Figure 2 C) Combining molecular biological identification, for strains 16S rRNA Gene amplification by PCR, sequencing, and BLAST homology comparison showed that this strain is similar to *Pseudomonas syringae* (…). Pseudomonas syringae The related strains showed extremely high sequence similarity, and phylogenetic analysis indicated that they clustered in the same branch with various pathogenic species of *Pseudomonas syringae*, such as... Figure 3 As shown. Based on morphological characteristics and molecular identification results, the isolated pathogenic bacterium was confirmed to be... Pseudomonas syringae .

[0031] Experimental results show that the identification results of the method provided by this invention are consistent with those of traditional methods, indicating that the method provided by this invention is suitable for PCR detection and molecular identification of strains.

[0032] Example 2

[0033] Following the method provided in Example 1, bacterial DNA was extracted from diseased leaf and lesion tissues of cucumber, sesame, green bean, soybean-E, rice, sorghum, soybean-G, and soybean-D collected from multiple regions across the country. The extracted DNA was then subjected to PCR detection. The results are as follows: Figure 5 As shown in the figure. The results show that all tested samples successfully amplified the corresponding target bands, which were clear, highly specific, and without obvious extraneous bands.

[0034] Preliminary detection of bacterial DNA in the diseased leaves of the above-mentioned plants was performed using gel electrophoresis. 16S rRNA The size and purity of the target band obtained by gene PCR amplification were determined, and the sequence was then sequenced. The sequenced sequences were entered into the NCBI website for BLAST homology comparison to obtain similarity. Based on the results, a phylogenetic tree was constructed, and the results are as follows: Figure 6 As shown. The results showed that cucumber, sesame, green beans, and soybean-E were associated with *Pseudomonas syringae* (…). Pseudomonas syringae The similarity is high, with rice, sorghum, and soybean-G showing similarities to Pantotheca ( ). Pantoea sp. Soybean-D and Xanthomonas oryzae (soybean-D has a high similarity) are highly similar. Xanthomonas campestris LJBJ8 (PP651582.1) is on the same branch. This indicates that the method is applicable to the rapid on-site extraction of pathogenic DNA from bacterial diseases of different plants in different regions, and has good practical application value.

[0035] The following is an optimization process for the method provided in Example 1: I. Selection of lysis buffer 1. Reagent and Sample Preparation 1) Reagents In plant lesion samples, DNA accounts for a low proportion of the cellular components, while secondary metabolites such as proteins, polysaccharides, and polyphenols easily interfere with nucleic acid amplification. Therefore, extracting effectively amplifiable bacterial DNA is crucial for subsequent on-site testing, and the composition of the lysis buffer directly determines the extraction efficiency, nucleic acid quality, and operational safety. Six different lysis buffers were selected, and their extraction effects were tested. The selected DNA lysis buffers, their components, and concentrations are shown in Table 3.

[0036] Table 3. Lysis Buffers with Different Components

[0037] Prepare each lysis buffer according to the composition and concentration of the lysis buffer provided in Table 1. After filtration through a 0.22 μm filter membrane and sterilization, dispense 300 μL into centrifuge tubes. Add 0.1-0.2 g of sterile quartz sand to each tube, tighten the cap, and store at room temperature for later use.

[0038] 2) Sample Sample preparation was the same as in Example 1.

[0039] 2. Bacterial DNA extraction Diseased cucumber leaf lesion tissue was added to a centrifuge tube containing lysis buffer. The lesion tissue was thoroughly crushed in the centrifuge tube using a glass rod, and then placed in a water bath and boiled for 10 minutes to complete nucleic acid release, yielding the extract. The same treatment was performed on healthy cucumber leaf tissue.

[0040] 3. DNA concentration and purity detection To evaluate the extraction quality of DNA from cucumber leaves using the six lysis buffers listed in Table 1, the DNA mass concentration and A content in the extracts were determined using a BioSpec-nano nucleic acid and protein analyzer. 260 / A 280 and A 260 / A 230 The ratio was measured three times for each sample, and the results are shown in Table 4.

[0041] Table 4. Concentration and purity of cucumber leaf DNA extracted from lysis buffers with different components

[0042] As shown in Table 4, comparing the DNA concentration and purity extracted from diseased and healthy cucumber leaves using six different lysis buffers revealed that all lysis buffers had significantly higher DNA extraction efficiency from diseased leaves than from healthy leaves. The DNA concentration from diseased leaves ranged from 248.3 to 574.9 ng / μL, while that from healthy leaves was only 39.6 to 139.8 ng / μL. This is not only attributed to the significant increase in pathogenic bacterial biomass in the lesion tissue, but also closely related to the more complete destruction of plant cell structure and degradation of cell walls caused by pathogen infection, making it easier for the lysis buffer to penetrate and release nucleic acids. Among them, lysis buffer L2 extracted the highest concentration of DNA from the lesion tissue, reaching 574.9 ng / μL, which was significantly better than the other five lysis buffers in Table 4. However, its A 260 / A 280 The ratio of only 1.20 indicates that proteins or other impurities were extracted along with the lysate during the lysis process, while the A content of all lysates was low. 260 / A 230 The ratios were all below 1.0, which indicates that a certain amount of polysaccharide or phenolic impurities were extracted along with the pyrolysis process.

[0043] 4. PCR detection PCR detection was performed using a 10-fold diluted extract as a DNA template, and universal primers 27F / 1492R and Pseudomonas aeruginosa were used respectively. 16S Gene primers Ps-F / Ps-R and rpoD PCR detection was performed using gene primers rpoD-F / rpoD-R. The PCR detection procedure using universal primers 27F / 1492R was the same as in Example 1. 16S Gene primers Ps-F / Ps-R and rpoD The reaction system for PCR detection using gene primers rpoD-F / rpoD-R is the same as that in Table 2, and the primer sequences and reaction procedures used are as follows: Table 5 Pseudomonocytes 16S Genes and rpoD Nucleotide sequence of gene primer

[0044] Program: 94℃ pre-denaturation for 5 min; 95℃ denaturation for 60 s, 55℃ annealing for 60 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 10 min.

[0045] The amplification products were detected by 2% agarose gel electrophoresis, and the results are as follows: Figure 7 As shown. From Figure 7 It can be seen that DNA extracted from lesion tissue using only lysis buffer L2 was successfully amplified. 16S rRNA pseudomonocytes 16S and rpoDThe target band of the gene was not obtained with any of the other five lysis buffers. This indicates that not all DNA extracted with lysis buffers is suitable for subsequent PCR detection. Although the high sensitivity of PCR amplification and the tolerance of DNA polymerase to impurities mean that low-quality DNA obtained by slightly diluting the lysate can be used for PCR amplification, it was found that only the pathogenic DNA extracted from diseased leaves by alkaline lysis could be used for subsequent PCR after dilution. Other lysis buffers, although purer than those using alkaline lysis, did not produce bands on agarose gel electrophoresis. It is speculated that although other lysis buffers can remove impurities and improve DNA purity to some extent, their specific release efficiency for pathogenic bacteria in plant lesions is insufficient, or they may form stable complexes with inhibitors such as polyphenols and polysaccharides in the tissue during extraction, leading to co-precipitation or adsorption loss of genomic DNA. Ultimately, this results in an effective template concentration for PCR amplification that is too low, below the detection limit of agarose gel electrophoresis, thus making it impossible to observe a clear band. Although the DNA extracted with lysis buffer L2 did not achieve the desired purity, simple dilution effectively reduced the inhibitor concentration, relieving Taq enzyme inhibition and thus successfully achieving specific amplification of the pathogenic target gene. Therefore, further optimization of the lysis buffer will be conducted based on lysis buffer L2.

[0046] II. Optimization of NaOH Concentration and Extract Dilution Ratio 1. Reagent and Sample Preparation 1) Reagents Based on lysis buffer L2, the concentration of NaOH was optimized, and seven concentrations were set: 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L. The lysis buffer was prepared according to the corresponding concentration. The prepared lysis buffer was filtered through a 0.22 μm filter membrane, sterilized, and then dispensed into centrifuge tubes, 300 μL per tube. At the same time, 0.1~0.2 g of sterile quartz sand was added to each tube, the tube was tightly capped, and stored at room temperature for later use.

[0047] 2) Sample Sample preparation was the same as in Example 1.

[0048] 2. Bacterial DNA extraction DNA was extracted from cucumber leaves using seven different concentrations of NaOH solution, following the method described in Section I, "Selection of Lysis Buffer".

[0049] 3. DNA concentration and purity detection The concentration and purity of DNA in the extract were determined according to the test method provided in "I. Selection of Lysis Buffer". The results are shown in Table 6.

[0050] Table 6. Concentration and purity of DNA extracted from cucumber leaves using different concentrations of NaOH.

[0051] As shown in Table 6, with the increase of NaOH concentration, the concentration of DNA extracted from cucumber leaves by the lysis buffer gradually increased. The DNA extraction concentration in lesion tissue gradually increased from 527.5 ng / μL to 984.2 ng / μL, and the DNA extraction concentration in healthy tissue gradually increased from 60.6 ng / μL to 105.5 ng / μL. This indicates that the increase of NaOH concentration can effectively enhance the cell lysis ability, promote nucleic acid release, and thus increase the DNA extraction concentration.

[0052] 4. PCR detection The dilution factor of the extract was optimized, with five concentrations set: undiluted, diluted by 1, 3, 5, and 10 times. PCR detection was performed on the undiluted and diluted extracts according to the PCR detection method provided in Example 1. The results are as follows. Figure 8 As shown.

[0053] from Figure 8 It can be seen that the concentration of NaOH in the lysis buffer and the dilution factor of the extraction buffer significantly affect the PCR detection results. DNA extracted with 0.05, 0.1, and 0.2 mol / L NaOH, after 5-fold and 10-fold dilutions, all showed weak bands in PCR amplification, but the band brightness was insufficient. This indicates that within this concentration range, the extracted DNA template has a certain degree of amplifurity, but is still subject to some interference from impurities. A NaOH concentration of 0.01 mol / L is too low, resulting in incomplete lysis; while NaOH concentrations of 0.3, 0.4, and 0.5 mol / L are too high, although the extraction concentration is high, the amount of co-extracted impurities also increases significantly, inhibiting Taq enzyme activity, and no effective amplification bands were obtained. Undiluted DNA samples and those diluted 1-3 times also did not show clear bands, possibly due to impurity interference or unsuitable template concentration. The 0.05–0.2 mol / L NaOH concentration strikes a good balance between DNA extraction efficiency and amplifurity; dilution of the extraction buffer 5–10 times can effectively reduce impurity interference and achieve effective amplification.

[0054] III. Optimization of Lysis Buffer Composition and Bacterial DNA Extraction Process 1. Reagent and Sample Preparation 1) Reagents Based on the three NaOH concentrations screened above, SDS and EDTA were further added to the lysis buffer, and DNA was extracted from cucumber leaves using the six solutions listed in Table 7.

[0055] Table 7. Composition and Concentration of Lysis Buffer

[0056] Prepare lysis buffers according to the composition and concentration of each lysis buffer in Table 7. Filter the prepared lysis buffers through a 0.22 μm filter membrane, sterilize them, and dispense them into centrifuge tubes, 300 μL per tube. Tightly cap the tubes and store them at room temperature for later use.

[0057] 2) Sample Sample preparation was the same as in Example 1.

[0058] 2. Bacterial DNA extraction The bacterial DNA extraction process was further optimized by eliminating the step of grinding with quartz sand. Diseased cucumber leaf tissue was added to a centrifuge tube containing lysis buffer and then placed in a water bath for 10 minutes to release nucleic acids, yielding the extract. Healthy cucumber leaf tissue was treated in the same manner.

[0059] 3. DNA concentration and purity detection The concentration and purity of DNA in the extract were determined according to the test method provided in "I. Selection of Lysis Buffer". The results are shown in Table 8.

[0060] Table 8. Concentration and purity of cucumber leaf DNA extracted from optimized lysis buffer

[0061] As shown in Table 8, omitting the quartz sand grinding step and using only direct water bath treatment, the DNA concentration extracted from diseased cucumber leaves ranged from 350.5 to 469.2 ng / μL, while the DNA concentration extracted from healthy leaves ranged from 68.3 to 83 ng / μL. This indicates that a certain concentration of DNA template can still be obtained even without omitting the quartz sand grinding step. Under direct water bath treatment conditions, the DNA extraction efficiency was significantly improved by further adding 0.01% SDS and 0.1 mmol / L EDTA.

[0062] 4. PCR detection Using undiluted, 5-fold diluted, and 10-fold diluted extracts as DNA templates, PCR detection was performed according to the method provided in Example 1. The results are as follows. Figure 9 As shown.

[0063] like Figure 9 As shown, PCR detection of the undiluted extract yielded no bands, while dilutions of 5 and 10 times resulted in the amplification of relatively clear target bands. The best results were observed with L1 as the lysis buffer and a 5-fold dilution of the extract, producing clear bands with moderate brightness. Considering the potential for insufficient inoculum in diseased leaves during practical applications, this condition was ultimately chosen as the experimental protocol for subsequent experiments that do not rely on instruments.

[0064] To reduce reliance on traditional laboratory equipment for on-site testing, a thermos cup heating method is used instead of a traditional water bath to provide the temperature and environment required for DNA extraction, thus obtaining the solution provided in Example 1.

[0065] In summary, NaOH, as a strong alkaline reagent, disrupts the peptidoglycan structure of bacterial cell walls through high concentrations of OH⁻ ions, causing the cell walls to undergo saponification and dissolve. Simultaneously, the phospholipid bilayer of the cell membrane hydrolyzes under alkaline conditions, leading to membrane structure collapse and rapid release of intracellular nucleic acids. Plant pathogenic bacteria are mostly Gram-negative, with thinner peptidoglycan layers in their cell walls and outer membranes rich in lipopolysaccharides, making them more susceptible to lysis under alkaline conditions. This contrasts sharply with the tough cellulose cell walls of plant cells, which remain relatively stable during short-term alkaline treatment and do not release large amounts of plant genomic DNA, thus enabling selective extraction of pathogenic bacterial DNA. However, higher NaOH concentrations are not always better. Too low a concentration leads to incomplete lysis, while too high a concentration exacerbates the swelling of macromolecular polysaccharides and the oxidation of polyphenols in plant tissues, introducing more PCR inhibitors. Therefore, a balance needs to be struck between bacterial cell disruption efficiency and impurity control. SDS, as an anionic surfactant, can insert into the phospholipid bilayer of the bacterial cell membrane, forming a synergistic effect with the chemical lysis of NaOH. Simultaneously, it denatures proteins to form negatively charged complexes, reducing co-precipitation with DNA. EDTA, on the other hand, inhibits DNase activity by chelating metal ions, protecting DNA from degradation. Studies have found that when NaOH, SDS, and EDTA are used in combination to lyse pathogenic bacterial DNA in plant lesions, a high concentration of DNA extract can still be obtained even without grinding with quartz sand. Furthermore, using an insulated cup instead of a water bath resulted in successful DNA banding after dilution and PCR. This indicates that the optimized method can be used for rapid and effective DNA extraction and routine detection of pathogenic bacterial DNA from plant lesions in the field.

[0066] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A rapid field extraction method for DNA from lesions of plant bacterial diseases, characterized in that, The process includes the following steps: mixing diseased leaf and lesion tissue with lysis buffer, heating in a water bath in a thermos to release nucleic acids; the lysis buffer contains 0.05~0.2 mol / L NaOH.

2. The method for rapid field extraction of DNA from plant bacterial disease lesions according to claim 1, characterized in that, The lysis buffer comprises 0.05~0.2 mol / L NaOH, 0.01% SDS, and 0.1 mmol / L EDTA.

3. The method for rapid field extraction of DNA from plant bacterial disease lesions according to claim 1, characterized in that, The plants are cucumber, sesame, green bean, soybean-E, rice, sorghum, soybean-G or soybean-D.

4. The method for rapid field extraction of DNA from plant bacterial disease lesions according to claim 1, characterized in that, Bacterial diseases of plants are caused by *Pseudomonas syringae* (… Pseudomonas syringae Pantotheca ( ) Pantoea sp. ) or Xanthomonas oryzae ( Xanthomonas campestris ).

5. The method for rapid field extraction of DNA from plant bacterial disease lesions according to claim 1, characterized in that, The conditions for water bath heating inside the thermos are to heat at 95~100℃ for 10~15 minutes.

6. A method for rapid detection of DNA in lesions of plant bacterial diseases, characterized in that, Includes the following steps: S1. Nucleic acid release is completed using the method described in any one of claims 1 to 5 to obtain an extract; S2. Dilute the extract 5 to 10 times; S3. Use the diluted extract as a DNA template for PCR detection.

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