Method for detecting active watermelon bacterial fruit blotch germs by PMA-PCR (Polymethyl Methacrylate-Polymerase Chain Reaction) technology

Abstract

The invention discloses a method for detecting active watermelon bacterial fruit blotch bacteria by a PMA-PCR (Polymerase Chain Reaction) technology, which comprises the following steps: (1) collection and suspension of bacteria in a sample to be detected: suspending the sample with normal saline to obtain a bacterial suspension; (2) PMA treatment: adding propyl azide bromide (PMA) into the bacterial suspension; (3) blue light crosslinking: irradiating the bacterial suspension treated by the PMA under blue light to activate the covalent binding of the PMA and dead bacteria DNA (Deoxyribose Nucleic Acid); (4) DNA extraction: centrifugally collecting thalli, and extracting DNA by adopting a kit method; (5) PCR amplification: carrying out amplification by using a specific primer pair; and (6) result judgment: detecting an amplification product through agarose gel electrophoresis, wherein a target strip appears in an active bacterium sample, and no strip exists in a dead bacterium sample. The invention establishes a rapid, convenient, accurate and efficient method for detecting the active watermelon bacterial fruit blotch, so as to improve the disease detection capability and management efficiency.

Description

Technical Field

[0001] This invention relates to the field of plant bacteria detection, and in particular to a method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology. Background Technology

[0002] Bacterial fruit blotch (BFB) is caused by *Acidovorax citrulli*, a bacterium belonging to the phylum Proteobacteria, class Betaproteobacteria, order Burkholderiales, family Comamonadaceae, and genus *Acidovorax*. It is a quarantine disease that causes devastating damage to various cucurbitaceous crops, including watermelon and melon. Seed-borne infection is the primary source of infection. In the early stages, small water-soaked spots appear on the leaves, later developing into brown to black necrotic spots on the fruit surface. In severe cases, it leads to fruit rot and drop, causing huge yield losses and has become a major limiting factor in the global cucurbit industry.

[0003] Since its first report in my country in 1998, the disease has spread to major seed production areas such as Hainan, Shandong, Fujian, Hunan, and Guangdong. In severely affected areas, 80% of watermelons lose their commercial value. In 2007, the bacterial fruit spot pathogen of cucurbits was included in my country's list of quarantine pests for imported plants.

[0004] Distinguishing between viable and inactivated bacteria of watermelon bacterial fruit spot pathogens is crucial in all stages of agricultural development. Viable pathogens are key to disease transmission and development; differentiating between viable and inactivated pathogens improves the accuracy of disease detection, avoids false positives due to residual inactivated bacteria, and allows for a more precise assessment of the actual risk and severity of disease spread. In commercial seed production, seed companies typically rely on positive results from conventional PCR to sterilize seeds using methods such as hydrochloric acid acidification, sodium hypochlorite, or dry heat disinfection to completely inactivate the watermelon bacterial fruit spot pathogens. However, because conventional PCR cannot accurately distinguish between viable and inactivated pathogens, residual inactivated bacterial DNA can still be amplified and show a positive signal after treatment. Lacking the technical means to differentiate between viable and inactivated bacteria, these disinfected seeds, which have effectively lost their infectivity, are forced to be treated as "infected," downgraded, or destroyed, resulting in significant economic losses. Simultaneously, farmers struggle to obtain truly "zero-viable-bacteria" seeds, leaving the potential risk of disease in the field persistent, creating a continuous challenge for the industry.

[0005] In current industry practice, existing PCR technology has significant limitations. It cannot accurately distinguish between live and dead fruit spot pathogens. Dead bacterial DNA fragments can persist even after disinfection, leading to false positives and misjudgments. The direct consequence of misjudgment is over-control strategies. Even when dead bacteria dominate and live bacteria are extremely low, a sample may still be classified as high-risk, forcing the application of high-strength chemical agents. This not only wastes resources and increases production costs but also pollutes the environment and accelerates the evolution of antibiotic resistance in pathogens, creating a negative chain reaction. Traditional detection methods, such as the seedling growth method, can distinguish between live and dead bacteria, but they suffer from low sensitivity, long cycles, susceptibility to environmental conditions, and poor reproducibility, making them unsuitable for rapid detection. Therefore, accurate identification of live bacteria has become a key to breaking the deadlock for the sustainable and healthy development of the cucurbit industry. Summary of the Invention

[0006] In view of this, this invention proposes a method for detecting active bacterial fruit spot pathogens in watermelon using PMA-PCR technology. The combination of propidium azide bromide (PMA) and PCR technology (PMA-PCR) provides a new approach for detecting active pathogens. PMA-PCR technology is an improvement on the standard PCR detection method, based on the concepts of DNA-binding dyes and the intact cell membranes of active cells. The nucleic acid dye PMA inhibits the amplification of dead bacteria, thereby improving the detectability of live cells. Through screening of PMA concentration, photolysis time, bacterial concentration, etc., a highly efficient and accurate PMA-PCR system for identifying active fruit spot pathogens in watermelon seeds was established. The process from nucleic acid extraction to result output for a single sample can be completed within 3 hours, significantly shortening the detection cycle compared to conventional isolation and culture (3-5 days), achieving efficient and accurate specific detection of active pathogens. This provides technical support for early field warning, accurately determines the safety of seeds after disinfection, avoids losses due to misjudgments caused by non-infectious dead bacteria, and provides reliable technical support for seed quarantine and disease control, which is of great strategic significance for ensuring the safety of my country's watermelon industry.

[0007] This invention proposes a method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology, comprising the following steps: (1) Collection and suspension of bacteria in the sample to be tested: The sample is suspended in physiological saline to obtain a bacterial suspension; (2) PMA treatment: Add propidium azide bromide (PMA) to the bacterial suspension; (3) Blue light crosslinking: The bacterial suspension treated with PMA is placed under blue light to activate the covalent binding of PMA to the DNA of dead bacteria; (4) DNA extraction: Collect bacterial cells by centrifugation and extract DNA using a kit method; (5) PCR amplification: Amplification is performed using specific primer pairs; (6) Result determination: The amplification products were detected by agarose gel electrophoresis. The target band appeared in the live bacteria sample, while no band appeared in the dead bacteria sample.

[0008] In a further technical solution, in step (2) PMA treatment, the final concentration of PMA is 30 μM and the incubation time in the dark is 10 minutes.

[0009] In a further technical solution, in step (3) blue light crosslinking, the bacterial suspension treated with PMA is irradiated under blue light with a wavelength of 465±10 nm for 20 minutes to activate the covalent binding of PMA with the DNA of dead bacteria; the blue light irradiation is carried out under ice bath conditions, and the light source is 15-25 cm away from the sample.

[0010] In a further technical solution, in the PCR amplification step (5), the specific primer sequences are: WFB1: 5'-GACCAGCCACACTGGGAC-3', WFB2: 5'-CTGCCGTACTCCAGCGAT-3'.

[0011] In a further technical solution, during the collection and suspension of bacteria in the sample to be tested in step (1), the concentration of the bacterial suspension is ≥10. 3 CFU / mL.

[0012] In a further technical solution, in step (1) of collecting and suspending bacteria in the sample to be tested, the verification of dead bacteria is achieved by inactivating them in a 100°C water bath for 10 minutes and then coating them onto a plate, with no colony growth observed.

[0013] The present invention provides a method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology, which has the following advantages over existing technologies: 1. This invention provides a new approach for detecting live pathogens by combining propidium azide bromide (PMA) with PCR technology (PMA-PCR). PMA-PCR is an improvement on the standard PCR detection method, based on the concepts of DNA-binding dyes and the intact cell membranes of live cells. The nucleic acid dye PMA inhibits the amplification of dead bacteria, thereby improving the detectability of live cells. Through screening of PMA concentration, photolysis time, bacterial concentration, etc., a highly efficient and accurate PMA-PCR system for identifying live watermelon seed blotch pathogens was established. Experimental results show that when the working concentration of PMA is 30 μM, incubation for 5 min in the dark followed by exposure for 20 min, this method can inhibit the DNA amplification of 10⁴ CFU / mL of dead watermelon bacterial blotch pathogens without affecting the amplification of live bacteria. This indicates that the established PMA-PCR detection method can effectively distinguish between live and dead watermelon bacterial blotch pathogens. 2. The PMA-PCR detection method established in this invention has strong specificity and high sensitivity, with a detection limit of 10³ CFU / mL. This invention can accurately distinguish between live and dead bacteria in samples, significantly reducing false positives. It is of great significance for the early diagnosis of watermelon fruit spot disease, import and export quarantine, and understanding the source of infection, transmission route, and epidemic pattern of the pathogen, as well as for formulating scientific and efficient prevention and control measures. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the results of adding different concentrations of PMA to the 104 CFU / mL bacterial suspension in this invention; Figure 2 This is a diagram showing the results of selecting the optimal exposure time in this invention; Figure 3 This is a graph showing the sensitivity test results of the PMA-PCR detection method in this invention. Detailed Implementation

[0015] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] like Figure 1 , Figure 2 , Figure 3 As shown, the present invention preferably provides a method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology, comprising the following steps: (1) Collection and suspension of bacteria in the test sample: Seed sample: Add 10 g of crushed seeds to 100 mL of physiological saline, shake at room temperature for 4 h, soak overnight at 4℃, centrifuge the soaking solution at 1000 rpm for 1 min, remove the precipitate, centrifuge the supernatant at 10000 rpm for 10 min, discard the supernatant, and collect the precipitate. Resuspend in sterile water to 1×10⁻⁶. 4 CFU / mL, take 1mL of bacterial culture and add it to a centrifuge tube for later use; Dead bacteria control: Inactivated in a 100℃ water bath for 10 min, and no colony growth was verified by plating; (2) PMA treatment: Add PMA working solution (final concentration 30 μmol / L, PMA, Biotium, USA) to the above centrifuge tubes, mix thoroughly, and incubate at room temperature in the dark for 10 min; (3) Blue light crosslinking: Place the sample tube on ice and use a blue LED light source with a wavelength of 465-475 nm to activate the covalent binding of PMA with dead bacterial DNA; keep the light source 15 cm away from the sample and irradiate for 20 min (shake gently every 5 min) to crosslink PMA with dead bacterial DNA. (4) DNA extraction: DNA was extracted using the Vazyme FastPure Bacteria DNA Isolation Mini Kit. (5) PCR amplification: Reaction system: Primer sequences: WFB1: 5'-GACCAGCCACACTGGGAC-3′ WFB2: 5′-CTGCCGTACTCCAGCGAT-3′ (360bp) Amplification procedure: Pre-denaturation: 94 ℃, 2 min Denaturation: 95℃, 20s Annealing: 55℃, 20s Extension: 72℃, 35s Number of cycles: 35 × Extension: 72℃, 7min (6) Result determination: The amplification products were detected by agarose gel electrophoresis. The target band appeared in the live bacteria sample, while no band appeared in the dead bacteria sample. Example

[0017] Optimization of PMA optimal concentration: A bacterial suspension with a plate count of 1x104 CFU / mL was selected. Ten 1mL aliquots of the bacterial suspension were taken, and five of them were placed in a 100℃ water bath for 10 min to prepare a dead bacterial suspension. After being spread on KB plates and incubated for 72 h, the absence of colony growth confirmed that the bacteria were dead.

[0018] Different amounts of PMA were added to 1x10⁴ CFU / mL dead bacterial suspensions and live bacterial suspensions to achieve final concentrations of 0 μM, 10 μM, 20 μM, 30 μM, and 45 μM, respectively. The suspensions were incubated in the dark for 10 min, then exposed to light for 20 min. DNA was extracted and PCR was performed. The results are as follows: Figure 1 As shown, the bands gradually weakened as the concentration of the added PMA working solution increased. When the final concentration of added PMA reached 30 μM, the bands in the dead bacteria group completely disappeared, and the brightness of the bands in the live bacteria group was consistent with that in the untreated group. Therefore, a final PMA concentration of 30 μM was selected as the optimal concentration for subsequent experiments. Example

[0019] Optimization of PMA optimal exposure time: A bacterial suspension with a plate count of 1x10⁴ CFU / ml was selected. Eight 1mL aliquots of the suspension were taken, four of which were used to prepare dead bacterial suspensions. PMA working solution was added and mixed to a final concentration of 30μM. The suspensions were incubated in the dark for 10 min, and then exposed to light for 5 min, 15 min, 20 min, and 30 min, respectively, before PCR reactions were performed. The results are as follows. Figure 2 As shown, the shortest time for the dead bacteria group to be completely band-free was 20 minutes. Therefore, the PMA exposure time of 20 minutes was selected as the optimal exposure time for subsequent experiments. Example

[0020] Sensitivity detection of PMA-PCR for bacterial fruit spot pathogens of watermelon: Viable *Pseudomonas aeruginosa* bacteria, a serially diluted fungus of 10⁷ CFU / mL, were detected using PMA-PCR. PMA working solution was added and mixed to a final concentration of 30 μM. The mixture was incubated in the dark for 10 min, then exposed to sunlight for 20 min before PCR. Results were obtained from... Figure 3 It can be seen that the lowest viable bacterial concentration at which a clearly visible band is formed, i.e., the detection limit, is 10. 3 CFU / mL.

[0021] In summary, the PMA-PCR detection method established in this invention can effectively distinguish between live and dead bacteria of watermelon bacterial fruit spot disease. Its optimal PMA concentration is 30 μM, the most suitable exposure time is 20 min, and its lower limit of detection is 10 μM. 3 CFU / mL.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology, characterized in that, Includes the following steps: (1) Collection and suspension of bacteria in the sample to be tested: The sample is suspended in physiological saline to obtain a bacterial suspension; (2) PMA treatment: Add propidium azide bromide (PMA) to the bacterial suspension; (3) Blue light crosslinking: The bacterial suspension treated with PMA is placed under blue light to activate the covalent binding of PMA to the DNA of dead bacteria; (4) DNA extraction: Collect bacterial cells by centrifugation and extract DNA using a kit method; (5) PCR amplification: Amplification was performed using specific primer pairs; (6) Result determination: The amplification products were detected by agarose gel electrophoresis. The target band appeared in the live bacteria sample, while no band appeared in the dead bacteria sample.

2. The method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology according to claim 1, characterized in that: In step (2) PMA treatment, the final concentration of PMA is 30 μM, and the incubation time in the dark is 10 minutes.

3. The method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology according to claim 1, characterized in that: In step (3) blue light crosslinking, the bacterial suspension treated with PMA is irradiated under blue light with a wavelength of 465±10 nm for 20 minutes to activate the covalent binding of PMA with the DNA of dead bacteria; the blue light irradiation is carried out under ice bath conditions, and the light source is 15-25 cm away from the sample.

4. The method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology according to claim 1, characterized in that: In step (5) of PCR amplification, the specific primer sequences are as follows: WFB1: 5'-GACCAGCCACACTGGGAC-3', WFB2: 5′-CTGCCGTACTCCAGCGAT-3′.

5. The method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology according to claim 1, characterized in that: In step (1), during the collection and suspension of bacteria in the sample to be tested, the bacterial suspension concentration is ≥10. 3 CFU / mL.

6. The method for detecting active bacterial fruit spot pathogens of watermelon using PMA-PCR technology according to claim 5, characterized in that: In step (1), the collection and suspension of bacteria in the sample to be tested, the verification of dead bacteria was achieved by inactivating them in a 100°C water bath for 10 minutes and then coating them onto a plate, with no colony growth observed.

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