A rapid detection method for anthrax in ancient and famous trees

Abstract

The present invention aims to solve the problems of low anthrax detection efficiency, inaccurate detection results, high detection cost, complicated and time-consuming operation in ancient and famous trees, and provides a rapid detection method for anthrax in ancient and famous trees. By quickly and effectively culturing anthrax in vitro, the detection rate in the early stage of anthrax infection is greatly improved; by designing a special culture medium, oscillating culture, two bacterial enrichment treatments, ultrasonic treatment, acidification treatment, blue light irradiation treatment and other means, the in vitro culture efficiency of anthrax in ancient and famous trees is greatly improved, and through the final Congo red staining, anthrax can be accurately and effectively detected.

Description

Technical Field

[0001] The present invention relates to the technical field of tree fungus detection, and in particular to a method for rapid detection of anthrax fungi in ancient and famous trees. Background Art

[0002] my country is rich in ancient and valuable trees, particularly in Guangdong and Hainan. However, in recent years, many have begun to age and falter, their immunity weakening and their ability to resist viruses weakening. Coupled with the severity of pests and diseases, these trees are prone to withering, withering, balding, yellowing leaves, and even death. Ancient trees are like a person in their seventies: frail, with weakened immunity and a weaker ability to resist viruses, they are easily weakened by pests and diseases. Effective monitoring is essential for the timely and effective protection of these ancient and valuable trees.

[0003] Many fungi in the genera Bivalvia, Aspergillus, and Colletotrichum can cause anthrax. Among them, Colletotrichum is the most common pathogen causing plant anthrax and is considered the eighth most important fungal pathogen worldwide. Colletotrichum has a wide host range, including ancient and valuable trees, many food crops, cash crops, and landscape plants. For these rare and valuable trees, accurate identification of Colletotrichum is crucial for the prevention and control of anthrax and for further research. Before the 1990s, the classification and identification of Colletotrichum relied primarily on morphological characteristics such as the shape and size of conidia and appressoria, and the presence and morphology of bristles, sclerotia, and chlamydospores. However, due to the instability of pure culture characteristics, spore and appressorium morphology, and their significant influence on the host and environment, similar or identical morphological features are often observed, making accurate identification based solely on morphology difficult. With the advancement of molecular biology, molecular identification techniques based on the internal transcribed spacer region of ribosomal DNA have been introduced for the classification and identification of Colletotrichum. However, ITS sequence analysis remains insufficient in systematic studies of this genus, failing to effectively distinguish closely related species. Subsequently, taxonomic identification methods based on combined multi-gene sequence analysis, supplemented by ecological, geographic, and morphological data, have become increasingly common in systematic studies of the genus Colletotrichum. Although molecular biological detection techniques have been widely applied in recent years, significantly advancing rapid detection of Colletotrichum, these techniques require expensive equipment, demanding technical expertise, and complex procedures, making them difficult to implement at grassroots testing institutions.

[0004] Anthracnose of ancient and famous trees has a long incubation period and is not easy to be discovered. Under suitable climatic conditions, it can break out into a disaster in a very short time, making prediction difficult. Early detection technologies for pathogenic fungi include traditional quantitative methods, enzyme-linked immunosorbent assay (ELISA), molecular detection technology, etc. These early detection technologies have relatively poor specificity, low sensitivity, and are easily interfered with by plant factors. In addition, the reagents and consumables are expensive, and the operation is cumbersome and time-consuming. Hainan University has invented a staining method for detecting pathogenic fungi on leaves (CN202010463836.0), which solves the problems of unclear staining levels, blurred images, and long operation time in current conventional fungal staining research, and achieves rapid and efficient staining of pathogens. However, it directly stains leaves cut into blocks. When the number of anthrax fungi is small in the early stage of infection, the detection accuracy cannot be guaranteed. Moreover, it requires fixation, decolorization, and vacuum filtration before staining, and the operation is relatively cumbersome. Therefore, there is an urgent need for a method for detecting anthrax fungi in ancient and famous trees with accurate test results, high detection efficiency, and fast, simple, and economical detection. Summary of the Invention

[0005] The present invention aims to solve the problems of low anthrax detection efficiency, inaccurate detection results, high detection cost, complicated and time-consuming operation in ancient and famous trees, and provides a rapid detection method for anthrax in ancient and famous trees. By quickly and effectively culturing anthrax in vitro, the detection rate in the early stage of anthrax infection is greatly improved; by designing a special culture medium, oscillating culture, two bacterial enrichment treatments, ultrasonic treatment, acidification treatment, blue light irradiation treatment and other means, the in vitro culture efficiency of anthrax in ancient and famous trees is greatly improved, and through the final Congo red staining, anthrax can be accurately and effectively detected.

[0006] The present invention adopts a novel in vitro culture method of anthrax fungi and a simple, effective and harmless staining method, which improves the detection efficiency and accuracy of anthrax fungi in ancient and famous trees, saves detection costs, and is very beneficial to the timely prevention and treatment of anthrax disease in ancient and famous trees.

[0007] The present invention is achieved through the following technical solutions:

[0008] A rapid detection method for anthrax in ancient and valuable trees comprises the following steps:

[0009] (1) Prepare a special culture medium: take 250g of peeled potatoes, crush them, add 1000ml of distilled water, 35g of glucose, 50g of sawdust, 20g of wheat bran, 30g of agar, and 10g of calcium carbonate, adjust the pH to 7.5, sterilize at 121℃ for 30min, and cool to room temperature for use;

[0010] (2) Oscillating culture: Take two abnormal leaves of ancient and famous trees, crush them, and place them in a culture bottle containing 1L of the above-mentioned special culture medium. Oscillating culture is carried out at 500 rpm for 6 hours at a temperature of 38°C.

[0011] (3) First enrichment: Add 100 ml of enrichment nutrient into the culture bottle and incubate at 30°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 18% peptone, 5% Jerusalem artichoke powder, 1% oligohexose, 1% sodium pyruvate, 0.5% pyridoxine hydrochloride, 1.2% ammonium ferric citrate, 1.5% potassium chloride, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use;

[0012] (4) Ultrasonic treatment: The culture mixture was treated with ultrasound at a frequency of 10 kHz for 3 min;

[0013] (5) Acidification: Add 10 ml of acetic acid to the culture mixture in the culture bottle, shake well, and incubate for 1 hour;

[0014] (6) Secondary enrichment: add 60 ml of enrichment nutrient to the culture bottle and culture at 33°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 12% yeast powder, 3% Jerusalem artichoke powder, 1% sodium selenite, 0.1% acetylcholine, 1.5% lithium chloride, 0.5% disodium hydrogen phosphate, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use;

[0015] (7) Blue light irradiation: Irradiate the culture mixture with a blue light lamp for 20 minutes. The wavelength of the blue light lamp used is 400-450 nm and the light intensity is 800-900 lx.

[0016] (8) Dark treatment: After irradiation with blue light, the culture flask was placed in complete darkness for 20 min;

[0017] (9) pH adjustment: Take out the culture bottle after dark treatment and adjust the pH to 7.5 with 10% hydrochloric acid solution;

[0018] (10) Staining: Take a small amount of the culture mixture and place it in a clean culture dish. Pass a low flame under the culture dish 3-4 times to evaporate the water in the culture dish. After cooling, add an appropriate amount of Congo red dye to the culture dish and place it at room temperature away from light for 30 minutes. Then pour out the dye and gently wash the culture dish with distilled water several times until there is no dye color in the water. Use absorbent paper to absorb excess water and observe the culture dish under a microscope. If a transparent circle appears, it is concluded that the ancient tree is infected with anthrax.

[0019] The present invention selects a special culture medium for anthrax bacteria containing potatoes, sawdust and wheat bran, which not only has a low production cost but also greatly improves the in vitro culture speed of anthrax bacteria.

[0020] The shaking culture in the first step of the present invention is not only conducive to the rapid adaptation of anthrax bacteria to the culture environment, but also conducive to the rapid and effective absorption of nutrients by anthrax bacteria, and plays a role in increasing bacteria to a certain extent.

[0021] The present invention selects two enrichment treatments and also adopts two different enrichment nutrients. The first enrichment nutrient contains peptone, which can play a greater enrichment role in the early stage of anthrax bacteria cultivation. At the same time, the synergistic effect of pyridoxine hydrochloride and ammonium ferric citrate also greatly increases the growth rate and metabolic rate of anthrax bacteria. The second enrichment nutrient contains yeast powder. After the anthrax bacteria have undergone the first enrichment and related treatments, it can consolidate and strengthen the proliferation and metabolism of anthrax bacteria. At the same time, the synergistic effect of acetylcholine and lithium chloride promotes the absorption of nutrients by anthrax bacteria. Both enrichment treatments use Jerusalem artichoke powder. For anthrax bacteria, Jerusalem artichoke powder is a good active protective agent, ensuring the activity of anthrax bacteria during cultivation.

[0022] In the interval between the two enrichment treatments, the present invention performs ultrasonic treatment and acidification. The 3-minute, 10 kHz ultrasonic treatment further enhances the first enrichment effect and makes the culture mixture more uniform. The subsequent acidification helps restore the anthrax bacteria and prepares them for the second enrichment treatment.

[0023] The present invention performs blue light irradiation, darkness treatment, and pH adjustment after the second enrichment treatment. The blue light irradiation helps stimulate the activity of anthrax bacteria after the enrichment is complete, promoting their growth and reproduction to a certain extent. The darkness treatment allows the anthrax bacteria to rest briefly after their rapid growth, facilitating the subsequent dyeing process. The pH adjustment before dyeing ensures dyeing efficiency and accuracy.

[0024] The present invention greatly improves the in vitro culture efficiency of anthrax in ancient and famous trees by designing a special culture medium, oscillating culture, two bacterial enrichment treatments, ultrasonic treatment, acidification treatment, blue light irradiation treatment, dark treatment and pH adjustment, and accurately and effectively detects anthrax in ancient and famous trees through the final Congo red staining. DETAILED DESCRIPTION

[0025] 1. A rapid detection method for anthrax in ancient and valuable trees, comprising the following steps:

[0026] (1) Prepare a special culture medium: take 250g of peeled potatoes, crush them, add 1000ml of distilled water, 35g of glucose, 50g of sawdust, 20g of wheat bran, 30g of agar, and 10g of calcium carbonate, adjust the pH to 7.5, sterilize at 121℃ for 30min, and cool to room temperature for use;

[0027] (2) Oscillating culture: Take two abnormal leaves of ancient and famous trees, crush them, and place them in a culture bottle containing 1L of the above-mentioned special culture medium. Oscillating culture is carried out at 500 rpm for 6 hours at a temperature of 38°C.

[0028] (3) First enrichment: Add 100 ml of enrichment nutrient into the culture bottle and incubate at 30°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 18% peptone, 5% Jerusalem artichoke powder, 1% oligohexose, 1% sodium pyruvate, 0.5% pyridoxine hydrochloride, 1.2% ammonium ferric citrate, 1.5% potassium chloride, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use;

[0029] (4) Ultrasonic treatment: The culture mixture was treated with ultrasound at a frequency of 10 kHz for 3 min;

[0030] (5) Acidification: Add 10 ml of acetic acid to the culture mixture in the culture bottle, shake well, and incubate for 1 hour;

[0031] (6) Secondary enrichment: add 60 ml of enrichment nutrient to the culture bottle and culture at 33°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 12% yeast powder, 3% Jerusalem artichoke powder, 1% sodium selenite, 0.1% acetylcholine, 1.5% lithium chloride, 0.5% disodium hydrogen phosphate, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use;

[0032] (7) Blue light irradiation: Irradiate the culture mixture with a blue light lamp for 20 minutes. The wavelength of the blue light lamp used is 400-450 nm and the light intensity is 800-900 lx.

[0033] (8) Dark treatment: After irradiation with blue light, the culture flask was placed in complete darkness for 20 min;

[0034] (9) pH adjustment: Take out the culture flask after dark treatment and adjust the pH to 7.5 with 10% hydrochloric acid;

[0035] (10) Staining: Take a small amount of the culture mixture and place it in a clean culture dish. Pass a low flame under the culture dish 3-4 times to evaporate the water in the culture dish. After cooling, add an appropriate amount of Congo red dye to the culture dish and place it at room temperature away from light for 30 minutes. Then pour out the dye and gently wash the culture dish with distilled water several times until there is no dye color in the water. Use absorbent paper to absorb excess water and observe the culture dish under a microscope. If a transparent circle appears, it is concluded that the ancient tree is infected with anthrax.

[0036] 2. Oscillating Culture of Anthrax

[0037] The present invention performs an oscillation culture on the anthrax bacteria in the leaves before the bacterial growth, which is not only conducive to the rapid use of the culture environment by the anthrax bacteria to absorb the influence, but also can play a role in pre-enrichment. Therefore, the oscillation culture before the anthrax bacteria is enriched is very necessary. As for the optimization of the oscillation speed, time and culture temperature, combined with the growth characteristics of anthrax bacteria in the leaves, the experimenters conducted multiple groups of comparative experiments, setting oscillation speeds of 200rpm, 500rpm, 800rpm, 1000rpm, 1200rpm, 1500rpm, and 2000rpm and culture times of 2, 4, 6, 8, 10, and 12 hours, as well as culture temperatures of 30°C, 32°C, 34°C, 36°C, 38°C, and 40°C. According to the experimental results, the oscillation culture at 500rpm for 6 hours and a culture temperature of 38°C were finally selected.

[0038] 3. Bacterial enrichment treatment of anthrax and selection of enrichment nutrients

[0039] In order to accelerate the in vitro culture of anthrax bacteria, the present invention adopts a secondary enrichment method. After a large number of screening and comparative tests, the researchers selected the ingredients of the two enrichment nutrients. The first enrichment nutrient contains peptone, which can play a significant enrichment role in the early stage of anthrax bacteria cultivation. At the same time, the synergistic effect of pyridoxine hydrochloride and ammonium ferric citrate also greatly increases the growth rate and metabolic rate of anthrax bacteria. The additives oligohexose, sodium pyruvate, and potassium chloride play a harmonizing role in the entire enrichment nutrient, reducing the stress response of anthrax bacteria to the enrichment nutrient. The second enrichment nutrient contains yeast powder, which can consolidate and strengthen the proliferation and metabolism of anthrax bacteria after the anthrax bacteria have undergone the first enrichment and related treatments. At the same time, the synergistic effect of acetylcholine and lithium chloride promotes the absorption of nutrients by anthrax bacteria. Sodium selenite and disodium hydrogen phosphate play a harmonizing role in the entire enrichment nutrient, reducing the stress response of anthrax bacteria to the enrichment nutrient. Both enrichment treatments used Jerusalem artichoke powder, a highly effective active preservative for anthrax bacteria, ensuring its viability during cultivation. Through extensive creative effort, the researchers determined the composition of the two enrichment nutrient solutions. Optimizing their ratios, they conducted extensive comparative testing and determined the specific formulas for the two solutions based on optimal enrichment results. The formula for the first enrichment treatment was: by weight, 18% peptone, 5% Jerusalem artichoke powder, 1% hexooligosaccharides, 1% sodium pyruvate, 0.5% pyridoxine hydrochloride, 1.2% ammonium ferric citrate, 1.5% potassium chloride, and the remainder was distilled water. The formula for the second enrichment treatment was: by weight, 12% yeast extract, 3% Jerusalem artichoke powder, 1% sodium selenite, 0.1% acetylcholine, 1.5% lithium chloride, 0.5% disodium hydrogen phosphate, and the remainder was distilled water.

[0040] 4. Exploration of Ultrasonic Treatment and Acidification Treatment on Anthrax Culture

[0041] Anthrax bacteria rapidly multiply after the first enrichment. This can lead to uneven nutrition distribution in the culture medium, which can also affect the vitality of the bacteria. Therefore, consolidation is necessary after the initial enrichment to ensure uniform nutrition and vitality. The researchers performed ultrasonic treatment and acidification on the culture mixture after the initial enrichment. Ultrasonication further enhanced the initial enrichment effect and made the culture mixture more uniform. The subsequent acidification helped restore the vitality of the anthrax bacteria, preparing them for the second enrichment.

[0042] As for the optimization of the frequency and treatment time of the ultrasound used, the researchers conducted a large number of comparative experiments, setting frequency gradients of 10, 20, 30, 40, 50, and 60 kHz and treatment time gradients of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 minutes respectively. Based on the experimental results, they finally selected the frequency of 10 kHz with the greatest impact for 3 minutes of treatment.

[0043] As for the optimization of the concentration and time of acetic acid treatment, the researchers conducted a large number of comparative experiments, setting concentration gradients of 5ml, 10ml, 15ml, 20ml, 25ml, and 30ml (per liter of culture medium mixture) and treatment time gradients of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 hours. Based on the experimental results, they finally selected the frequency with the greatest impact, 10ml treatment for 1 hour.

[0044] 5. Effect of blue light irradiation on the proliferation of anthrax bacteria

[0045] Anthrax bacteria will continue to rapidly reproduce after the second enrichment. Excessive reproduction can affect their vitality due to various factors. Therefore, it is important to maintain the vitality of the anthrax bacteria even after the second enrichment. Blue light irradiation helps stimulate the vitality of the anthrax bacteria after the enrichment is complete, and can also promote its growth to a certain extent.

[0046] As for the optimization of the light intensity and time of blue light irradiation, the researchers conducted a large number of comparative experiments, setting light intensity gradients of 700-800lx, 800-900lx, 900-1000lx, 1000-1100lx, 1100-1200lx, and 1200-1300lx and treatment time gradients of 10, 20, 30, 40, 50, and 60 minutes. Based on the experimental results, they finally selected the light intensity of 800-900lx for 20 minutes, which had the greatest impact.

[0047] VI. Effect of Dark Treatment on Post-enrichment Staining of Anthrax

[0048] After two rounds of bacterial growth and blue light irradiation, the metabolism of anthrax bacteria is in an extremely vigorous state. If staining is performed directly at this time, false positive results may occur. Therefore, the inventors darkened the culture bottles after blue light irradiation to ensure that the metabolism of anthrax bacteria is in a normal state and the accuracy of subsequent staining is guaranteed.

[0049] As for the optimization of the dark treatment time, the researchers set a treatment time gradient of 10, 20, 30, 40, 50, and 60 minutes respectively. The results showed that the metabolism of anthrax returned to normal after 20 minutes.

Claims

1. A rapid detection method for anthrax in ancient and valuable trees, characterized by: The following steps are involved: (1) Prepare a special culture medium: take 250g of peeled potatoes, crush them, add 1000ml of distilled water, 35g of glucose, 50g of sawdust, 20g of wheat bran, 30g of agar, and 10g of calcium carbonate, adjust the pH to 7.5, sterilize at 121℃ for 30min, and cool to room temperature for use; (2) Oscillating culture: Take two abnormal leaves of ancient and famous trees, crush them, and place them in a culture bottle containing 1L of the above-mentioned special culture medium. Oscillating culture is carried out at 500 rpm for 6 hours at a culture temperature of 38°C. (3) First enrichment: Add 100 ml of enrichment nutrient into the culture bottle and incubate at 30°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 18% peptone, 5% Jerusalem artichoke powder, 1% oligohexose, 1% sodium pyruvate, 0.5% pyridoxine hydrochloride, 1.2% ammonium ferric citrate, 1.5% potassium chloride, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use; (4) Ultrasonic treatment: The culture mixture was treated with ultrasound at a frequency of 10 kHz for 3 min; (5) Acidification: Add 10 ml of acetic acid to the culture mixture in the culture bottle, shake well, and incubate for 1 hour; (6) Secondary enrichment: add 60 ml of enrichment nutrient to the culture bottle and culture at 33°C for 2 h. The formula of the enrichment nutrient is as follows: by weight, 12% yeast powder, 3% Jerusalem artichoke powder, 1% sodium selenite, 0.1% acetylcholine, 1.5% lithium chloride, 0.5% disodium hydrogen phosphate, and the rest is distilled water; sterilize the enrichment nutrient at 121°C for 30 min before use; (7) Blue light irradiation: Irradiate the culture mixture with a blue light lamp for 20 min. The wavelength of the blue light lamp used is 400-450 nm and the light intensity is 800-900 lx. (8) Dark treatment: After irradiation with blue light, the culture flask was placed in complete darkness for 20 min; (9) pH adjustment: Take out the culture bottle after dark treatment and adjust its pH to 7.5 with 10% hydrochloric acid solution; (10) Staining: Take a small amount of the culture mixture and place it in a clean culture dish. Pass a low flame under the culture dish 3-4 times to evaporate the water in the culture dish. After cooling, add an appropriate amount of Congo red dye to the culture dish and place it at room temperature away from light for 30 minutes. Then pour out the dye and gently wash the culture dish with distilled water several times until there is no dye color in the water. Use absorbent paper to absorb excess water and observe the culture dish under a microscope. If a transparent circle appears, it is concluded that the ancient tree is infected with anthrax.