Mulberry sclerotinia sclerotiorum epidemic law analysis method based on spore capture

Abstract

The invention discloses a mulberry sclerotinia sclerotiorum epidemic law analysis method based on spore capture, which not only dynamically detects the dynamic change of bud (fruit) and soil mulberry sclerotinia sclerotiorum, but also utilizes a constant volume type spore capture instrument to capture a sample in the air, and utilizes a disposable swab cotton swab to collect a spore sample in the air. The high-throughput sequencing technology is used for germ detection, and compared with a traditional PCR technology, the detection sensitivity and accuracy are greatly improved. Meanwhile, meteorological factors, the change of the content of germs and the incidence rate of diseases are combined for analysis, it is found that about March 10 is the key period of germ infection, and the incidence rate has extremely high positive correlation with the relative humidity and the rainfall from the initial flowering stage to the full-bloom stage. The method provided by the invention provides a new method for dynamic detection of pathogens of both soil-borne diseases and airborne diseases, also provides a theoretical basis for disease prediction and early warning, and also provides a basis for selection of disease control periods.

Description

Technical Field

[0001] The invention relates to the field of mulberry sclerotinia pathogen detection, and in particular to a method for analyzing the epidemic regularity of mulberry sclerotinia pathogen based on spore capture. Background Art

[0002] Mulberry sclerotinia, also known as ginkgo disease, is common in planting areas with an incidence rate of 30% to 90%. In some places, the disease even leads to the complete loss of mulberry production, which is a devastating disease that harms the development of the fruit mulberry industry. Currently, there are three types of mulberry sclerotinia, mulberry sclerotinia and mulberry small sclerotinia, and the pathogens are Ciboria shiraiana, Scleromitrula shiraiana and Ciboria carunculoides. Wang Guoliang et al. also found Scleromitrula sp., Scleromitrularubicola and Synciboria ningpoensis in Ningbo; He Lei et al. reported that Phomamoricola can also cause mulberry sclerotinia, and its diseased fruit morphology is quite similar to that of mulberry fat sclerotinia; Hu Junhuan et al. found that Sclerotinia sclerotiorum can also cause mulberry sclerotinia at the Ningbo Tiangong Manor mulberry fruit base. Although the isolation and identification of Sclerotinia sclerotiorum have been reported, many scholars have failed to isolate Sclerotinia sclerotiorum from ascospores, sclerotia or diseased fruits. Sultana et al. (2013) first reported the existence of Sclerotinia carunculata in South Korea, but failed to isolate and culture the fungus on various culture media. Some studies believe that Sclerotinia sclerotiorum is an obligate parasite and cannot be cultured in vitro. Although Sclerotinia sclerotiorum can be isolated and cultured, the strain grows very slowly. In short, it is very difficult to detect pathogens using traditional methods.

[0003] Sclerotia is an important place for mulberry sclerotinia disease to overwinter. Sclerotia fall to the ground with the diseased mulberries. When the female mulberry flowers bloom the following year, the sclerotia in the soil germinate to form ascodiscs. Ascospores released by ascodiscs are the main source of initial infection of the disease. Ascospores can be spread through natural factors such as wind and rain to cause the occurrence of the disease, or they can be spread to distant places with air currents to cause the disease. Therefore, the transmission of mulberry sclerotinia disease includes soil transmission and air transmission. Because the disease currently relies mainly on two chemical agents, methyl thiophanate and carbendazim, for prevention and control, long-term use has caused sclerotinia disease to develop resistance to them. As the concentration of the drug continues to increase, the amount of pesticide residues also increases, and the quality of mulberry fruit and its edible safety cannot be guaranteed, which seriously affects the development of the subsequent processing industry of fruit mulberry.

[0004] The early symptoms of mulberry sclerotinia are not obvious. When the symptoms can be observed, it is almost impossible to effectively prevent and control it. At present, the detection of this disease mainly relies on the separation and purification of pathogens from diseased tissues, morphological and molecular biological identification and Koch's postulates verification. The whole process is long and difficult to isolate and culture the pathogens, which can easily delay the prevention and control. In recent years, with the development of molecular biology, a variety of molecular methods have been used for the detection of pathogens, such as conventional PCR, real-time fluorescent quantitative PCR (qPCR) and loop-mediated isothermal amplification technology. Compared with traditional pathogen detection methods, molecular detection methods have the advantages of objectivity, accuracy, rapidity and high throughput. However, these diagnostic technologies cannot achieve early warning before the disease spreads, and can only be detected after the pathogen infects. Environmental factors such as temperature and relative humidity can affect the number of pathogen spores that germinate, and will further affect the infection of pathogen spores to plants. As a bridge between pathogenic microorganisms and hosts, the environment plays a vital role in the occurrence and development of diseases. On this basis, constructing a correlation model between plant pathogens and environmental factors and providing early warning for plant pathogens can effectively prevent and reduce the occurrence of plant pathogens and ensure the healthy growth of plants.

[0005] Spore capture instrument is an instrument that can capture pathogen spores. It can not only monitor the dynamic changes in the number of spores in the air, but also reflect the laws of their spatiotemporal diffusion. If combined with crop growth conditions, disease dynamics, and meteorological factors such as temperature, humidity, and rainfall, the occurrence and prevalence of diseases can be explored. Glass slide capture is the earliest and most primitive spore capture method. A glass slide coated with an adhesive or a vessel containing a culture medium is placed in the field, and the spores to be captured are randomly adhered through air flow. However, because the capture glass slide or vessel is directly exposed to the air, it is easily disturbed by external factors (especially rainfall), and the capture is random and blind, which is not suitable for long-term dynamic monitoring. Therefore, this method has rarely been used in scientific research and production practice. The working principle of mobile spore capture instruments and portable spore capture instruments is mainly to capture spores by forming negative pressure inside themselves. They have good stability and accuracy and are suitable for situations where frequent movement or monitoring at different locations is required. The constant volume spore trap is currently the most widely used spore capture method, and the Burkard Hirst 7-day constant volume spore trap is the most commonly used in research. When the constant volume spore trap is working, the air drive device forms a negative pressure in the capture chamber, and the external air passes through the horizontal 2×14 mm slit-shaped air inlet at a speed of 10 liters / minute. The air sample also enters the sampler and hits the spore capture belt that continuously passes through the air inlet at a speed of 2 mm / hour. Each 48mm long strip represents a 24-hour air sample, and it can be collected continuously for seven days; because it has its own air drive device to provide it with power, it can ensure that the volume of air inhaled at every moment is fixed, and changes in external conditions are not easily affected. Through research, it has been shown that compared with previous spore capture methods, this method is more efficient in capturing spores.

[0006] The traditional method to determine the concentration of pathogen spores is microscopic counting, which is time-consuming and labor-intensive. Microscopic observation can easily cause visual fatigue, and the captured pathogen spores are mixed with a large number of other types of spores, dust or particles, which can easily lead to misjudgment. The professional requirements for microscopy personnel are also relatively strict. With the increasing status of molecular biology technology in the field of plant pathology research, PCR reaction can be used to achieve quantitative detection of the number of spores captured by the fixed-volume spore trap. However, the sensitivity of PCR detection is limited, and the pathogens need to accumulate to a certain concentration before they can be detected.

[0007] The present invention samples spores in buds (fruits), soil and air of two test bases in 2022, and uses high-throughput sequencing analysis to monitor changes in pathogen content in the above samples. It is found that pathogens were detected in the fruits and soil of the susceptible variety Yuezhen Da 10 (DS) and the more resistant fruit mulberry variety Taiwan Changguo Mulberry (CG) collected on February 18, and the pathogen content increased significantly around March 10, indicating that March 10 is a critical period for pathogen infection. Conventional PCR was used to detect the above samples, and only samples in the soil could be detected. This shows that the sensitivity and accuracy of this method are much higher than those of the conventional PCR method. The corrplot package in R (v4.41) was used to analyze the Pearson correlation between the number of sequence fragments of relevant meteorological data and three types of mulberry sclerotinia pathogens measured by microbial sequencing, and it was found that the changes in mulberry sclerotinia pathogens were highly correlated with various meteorological factors. From 2022 to 2024, Dynamet scientific research automatic weather stations were used to collect meteorological data. Correlation analysis between the incidence rate and meteorological factors from the early flowering period to the peak flowering period found that the incidence rate had a strong positive correlation with relative humidity and rainfall, with correlation coefficients of 0.99 and 0.98, respectively. In addition, this method can also monitor the changes in the mulberry brown spot pathogen that occurs during the same period. Summary of the invention

[0008] In view of this, the present invention provides a method for analyzing the epidemic pattern of mulberry sclerotinia pathogen based on spore capture.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture comprises the following steps:

[0011] Step 1: Sample collection

[0012] Before the fruit mulberry leaves turn green, the buds (fruits), fruits and soil of the disease-susceptible variety Yuezhenda 10 (DS) and the disease-resistant variety Taiwan Changguo mulberry (CG) were collected at the test site. At the same time, spores were captured at the test site using an automatic sampler, and spores on the spore capture strips were collected using a disposable swab.

[0013] Step 2: Sample DNA extraction

[0014] Extract the total genomic DNA of the microbial community, use agarose gel electrophoresis to detect the quality of the extracted genomic DNA, and determine the DNA concentration and purity;

[0015] Step 3: PCR amplification and sequencing library construction

[0016] Using the above-extracted DNA as a template, PCR amplification is performed, PCR products are recovered using agarose gel, the recovered products are purified using a DNA gel recovery and purification kit, and the recovered products are detected and quantified; the purified PCR products are library-built and sequenced;

[0017] Step 4: High-throughput sequencing data analysis

[0018] The double-end original sequencing sequences were quality controlled and spliced: the sequences after quality control splicing were clustered into operational taxonomic units (OTUs) and chimeras were removed. The relative abundance bar graph of the species-level community was analyzed, and the sequencing abundance of three types of mulberry sclerotinia fungi and Soft-coated fungi in the samples was analyzed;

[0019] Step 5: Analysis of pathogen epidemic patterns

[0020] The meteorological data collected were used to calculate the relevant meteorological data, and the daily average air temperature, daily average soil temperature, daily rainfall, daily average relative humidity, daily soil moisture content, daily average wind speed, and daily total solar radiation were obtained. The average value of the relevant meteorological data during the sampling period was calculated. The corrplot package in R (v4.41) was used to calculate the abundance values ​​of the three types of mulberry sclerotinia fungus sequence fragments and the Pearson coefficient with the above meteorological factors, and their correlation was analyzed, so as to analyze the factors affecting the epidemic spread of pathogens.

[0021] Step 6: Analysis of disease epidemic patterns

[0022] DS mulberry trees were selected at fixed locations for many years to conduct branch surveys, record the total number of fruits and diseased fruits on the branches, and calculate the incidence rate. The biological traits of DS were investigated, and the relationship between meteorological factors and disease incidence during the critical period of pathogen infection was analyzed in combination with high-throughput sequencing results.

[0023] Preferably, in step 1, the mulberry trees at test site 1 were planted in 2019 with limited root cultivation, with a row spacing of 3m×2m (114°33′E, 30°48′N); the field at test site 2 was planted in 2021, with a row spacing of 3m×2m, and is 500m away from the test base at site 1.

[0024] Preferably, in step 1, 3 DS and CG plants are selected at test points 1 and 2, 3 branches are selected from each mulberry tree, and 5 buds are collected from each branch; at the same time, the soil around each tree is collected, and five points are selected under each tree each time to collect soil samples of the 0-20 cm tillage layer, and debris such as gravel and plant roots are removed. Then, the soil around the three DS and CG plants is mixed and put into a ziplock bag, which is immediately sealed and taken back to the laboratory, and stored in a -80°C refrigerator after passing through a 1 mm sieve, and samples are taken every 7 days.

[0025] Preferably, in step 3, primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) are used for PCR amplification.

[0026] Preferably, in step 3, the PCR reaction system is: 4 μL of 5×TransStartFastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of 5 uM upstream primer, 0.8 μL of 5 μM downstream primer, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and the total volume is 20 μL;

[0027] The amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 27 cycles, then 72°C stable extension for 10 min, and finally storage at 4°C.

[0028] Preferably, in step 6, three DS mulberry trees are selected at a fixed location, three branches are randomly surveyed from each mulberry tree, each branch is surveyed for a length of 1 m, the total number of fruits and the number of diseased fruits on the 1 m branch are recorded, and the incidence rate is calculated as the number of diseased fruits / total number of fruits×100%.

[0029] Compared with the prior art, the present invention has achieved the following technical effects:

[0030] (1) Compared with traditional PCR technology, the present invention greatly improves the sensitivity and accuracy of monitoring;

[0031] (2) The present invention combines meteorological factors with changes in pathogen content and disease incidence and finds that March 10 is a critical period for pathogen infection, and the incidence rate has a strong positive correlation with relative humidity and rainfall from the early flowering stage to the peak flowering stage;

[0032] (3) The method of the present invention provides a new method for the dynamic monitoring of pathogens that are both soil-borne diseases and air-borne diseases, provides a theoretical basis for disease prediction and early warning, and also provides a basis for the selection of disease prevention and control periods. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The spore capture sampling diagrams of buds, fruits, soil and Burkard Hirst spore automatic sampler at different stages of the present invention;

[0034] Figure 2 The sequencing of microbial diversity of samples collected in 2022 for the present invention;

[0035] Among them, A. Species-level community relative abundance bar graph; B. Sequencing of three types of mulberry sclerotinia fungi in samples. C. Sequencing of Soft-coated fungi in samples;

[0036] Figure 3 This is a species-level heatmap of the microbial diversity sequencing community of samples collected in 2023 of the present invention;

[0037] Figure 4 This is the analysis diagram of the microbial diversity sequencing of brown spot pathogens collected in samples in 2022 of the present invention;

[0038] Figure 5 PCR detection of some samples of the present invention;

[0039] Among them, lanes 1-22: GDS1_412, TCG1_324, TCG1_41, TCG1_412, dian2_323, dian2_330, dian2_46, dian2_413, dian2_421, dian2_428, dian2_512, dian2_520, GDS1_218, GDS1_225, GDS1_34, GDS1_310, GDS1_317, TDS1_218, TDS1_225, TDS1_34, TDS1_310, TDS1_318;

[0040] Figure 6 The Pearson correlation diagram of the number of sequence fragments of three types of mulberry sclerotinia pathogens detected by meteorological data of the present invention and microbial sequencing;

[0041] Among them, TCAir: air temperature; RH: relative humidity; RG: rainfall; TCSoil: soil temperature; Smoist: soil moisture content; SR: total solar radiation; WS: wind speed;

[0042] Figure 7 This is a correlation analysis diagram of the incidence rate in 2022-2024 and meteorological factors from the initial flowering period to the full flowering period of the present invention;

[0043] Figure 8 This is a meteorological factor map during the field survey in 2022-2024 of the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Embodiment 1:

[0046] The present invention discloses a method for analyzing the epidemic regularity of mulberry sclerotinia sclerotiorum based on spore capture, comprising the following steps:

[0047] Step 1: Sample collection

[0048] From February 18 to April 12, 2022, two test sites were selected in Hubei Province for disease monitoring. The mulberry trees in test site 1 were planted in 2019 with limited root cultivation and a row spacing of 3m×2m (114°33′E, 30°48′N). The field in test site 2 was planted in 2021 with a row spacing of 3m×2m and 500m away from the test base of site 1. Three DS and CG plants were selected at both test sites 1 and 2, and three branches were selected from each mulberry tree, with five buds collected from each branch. At the same time, the soil around each tree was collected. Five points were selected under each tree each time to collect soil samples from the tillage layer (0-20cm), and debris such as gravel and plant roots were removed. Then, the soil around the three DS and CG plants was mixed and put into self-sealing bags, which were immediately sealed and taken back to the laboratory. After being sieved with 1mm, they were stored in a -80℃ refrigerator and samples were taken approximately every 7 days. The fruits and soil of DS and CG at test site 1 and the fruits and soil of DS at test site 2 were used for microbial diversity detection. At the same time, spores were captured at site 2 using a Burkard Hirst spore automatic sampler from March 15 to June 10, and spores captured in 7 days were collected using disposable swabs. On February 10, 2023, buds of DS and CG were collected from the above two test sites, and spores were captured at site 2 using a Burkard Hirst spore automatic sampler. The above collected samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for microbial diversity sequencing. Figure 1 shown.

[0049] Step 2: Sample DNA extraction

[0050] according to The total genomic DNA of the microbial community was extracted according to the instructions of soil DNA kit (Omega Bio-tek, Norcross, GA, US), the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000 (Thermo Scientific, USA).

[0051] Step 3: PCR amplification and sequencing library construction

[0052] The above-extracted DNA was used as a template, and primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were used for PCR amplification. The PCR reaction system was: 4 μL of 5×TransStartFastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStartFastPfu DNA polymerase, 10 ng of template DNA, and the total amount was 20 μL. The amplification program was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI 9700 model). PCR products were recovered using 2% agarose gel, purified using a DNA gel recovery and purification kit (PCR Clean-Up Kit, Yuhua, China), and detected and quantified using Qubit4.0 (Thermo Fisher Scientific, USA).

[0053] use The Rapid DNA-Seq Kit was used to construct a library for the purified PCR products: (1) linker ligation; (2) use magnetic beads to screen and remove linker self-linked fragments; (3) use PCR amplification to enrich the library template; (4) use magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using the Illumina Nextseq2000 platform.

[0054] Step 4: High-throughput sequencing data analysis

[0055] Fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software was used for quality control of the double-end raw sequencing sequences, and FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software was used for splicing: (1) The bases with a quality value of less than 20 at the end of the reads were filtered, and a 50 bp window was set. If the average quality value in the window was less than 20, the back-end bases were cut from the window, and the reads less than 50 bp after quality control were filtered, and the reads containing N bases were removed; (2) According to the overlap relationship between PE reads, the paired reads were spliced ​​(merged) into a sequence, and the minimum overlap length was 10 bp; (3) The maximum mismatch ratio allowed in the overlap region of the spliced ​​sequence was 0.2, and the non-compliant sequences were screened; (4) The samples were distinguished according to the barcodes and primers at the beginning and end of the sequence, and the sequence direction was adjusted. The number of mismatches allowed for the barcode was 0, and the maximum number of mismatches for the primers was 2. UPARSE v7.1 software (http: / / drive5.com / uparse / ) was used to cluster the quality control spliced ​​sequences into operational taxonomic units (OTUs) according to 97% similarity and remove chimeras. R (v4.41) was used to analyze the relative abundance bar graph of the species level community, and Excel was used to analyze the sequencing abundance of three types of Sclerotinia sclerotiorum and Soft-coated fungi in the samples.

[0056] Step 5: Routine PCR testing of disease samples

[0057] The PCR detection of some microbial diversity sequencing samples was performed using the previously designed C. carunculoides specific primers CCP-F (5'-AAATCCACGACAATCACGAAC-3') / CCP-R (5'-CGATGAATACCAACCCAAACA-3'). The total volume of the CR reaction system was 50 μL, and the reaction components were: 2×TaqMasterMix (Kangwei Century) 25 μL, 1 μL of 10 μM upstream and downstream primers, 1 μL of DNA, and 22 μL of double distilled water; the amplification reaction program was: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 45 s, a total of 35 cycles, and 72℃ extension for 10 min. After the PCR product was detected by 1.0% agarose gel electrophoresis, the gel imaging system analyzed the results.

[0058] Step 6: Analysis of disease epidemic patterns

[0059] From 2022 to 2024, meteorological data will be collected every year using the Dynamet scientific research-level automatic weather station set up near the experimental plot. The relevant meteorological data were calculated separately to obtain the daily average air temperature, daily average soil temperature, daily rainfall, daily average relative humidity, daily soil moisture content, daily average wind speed, daily total solar radiation and other related data. At the same time, the average value of the relevant meteorological data for the 7 days of the sampling period in 2022 was calculated. The corrplot package in R (v4.41) was used to calculate the Pearson coefficient of the correlation between the relevant meteorological data and the abundance of the sequence fragments of the three types of mulberry sclerotinia fungus sequenced by microbial sequencing.

[0060] From 2022 to 2024, on April 19th of each year, three DS mulberry trees at fixed locations were selected, and three branches of each mulberry tree were randomly surveyed. The total number of fruits and the number of diseased fruits on each branch of 1 m were recorded, and the incidence rate was calculated as the number of diseased fruits / total number of fruits × 100%. The corrplot package in R (v4.41) was used to analyze the relationship between meteorological factors and disease occurrence.

[0061] Example 2: Experimental results and analysis

[0062] 1. High-throughput sequencing analysis of spores in buds (fruits), soil and air

[0063] High-throughput sequencing analysis of spores in buds (fruits), soil and air in 2022 found that the abundance of fungi such as Alternaria, C. carunculoides and Cladosporium delicatulum was high. Mulberry sclerotinia was detected in CG and DS fruits collected on February 18. The pathogen content in Yuezhen Da 10 planted in the field of Point 1 increased significantly around March 10, and the pathogen content in Yuezhen Da 10 planted in the field of Point 2 also increased around March 10, and increased significantly on March 17. In the mulberry garden of Point 1, where the disease is severe every year, the content of the pathogen in the soil of the susceptible variety DS is significantly higher than that of the more resistant variety CG. The pathogen content in the soil of the newly built Point 2 mulberry garden is extremely low.

[0064] During the period of March 8-15, spores of mulberry sclerotinia were captured at both test points, indicating that the spore capture method is feasible. The amount of spore capture gradually increased between March 15-23 and March 23-30. Spores of mulberry sclerotinia were detected during the periods of April 6-13, April 13-21, April 21-28, April 28-12, and May 12-20. Spores of mulberry sclerotinia were also detected during May 20-16. Therefore, the spread of the pathogen may be different from the traditional understanding that the pathogen survives summer and winter with sclerotia after forming sclerotia, which deserves further study. Figure 2As shown. In the samples collected on February 10, 2023, the mulberry sclerotinia pathogen was only found in the CG buds at collection point 1, but the fungus was not captured in the air from February 10 to 17, as shown. Figure 3 As the reported sclerotinia pathogens of mulberry mainly involved pathogens of the order Helotiales, we further analyzed the changes of fungi of this order in the samples, which further indicated that C. shiraiana and C. carunculoides were the main pathogens in the two test sites.

[0065] In addition, mulberry brown spot disease was severe during the same period, with an incidence rate of up to 100%. Through the analysis of microbial diversity sequencing results, its pathogen Cercospora sp. was present in fruit, soil and air samples collected at two locations in 2022, and began to increase around February 25, and the content of pathogens in the air surged around March 15 to March 24. Figure 4 As shown. On February 10, 2023, the brown spot pathogen was found in the buds of CG and DS at points 1 and 2, and was also present in the spore capture strips, as shown Figure 3 This shows that brown spot pathogens can overwinter on leaves and trees, and dynamic monitoring of pathogens has laid a theoretical basis for disease prevention and control.

[0066] 2. Routine PCR testing of disease samples

[0067] like Figure 5 As shown, some samples were taken for conventional PCR testing using specific primers. The presence of pathogens was detected only in the soil samples collected from 2.18 to 3.18, indicating that the sensitivity of conventional PCR testing is low. High-throughput sequencing results greatly improve the accuracy of pathogen detection.

[0068] 3. Analysis of pathogen epidemic factors

[0069] like Figure 6As shown in the figure, the Pearson correlation analysis of the abundance of three types of mulberry sclerotinia pathogen sequences detected by microbial sequencing using the corrplot package in R (v4.41) showed that the content of the pathogen in the fruit of the disease-resistant variety GCG1 at point 1 was highly correlated with wind speed at 0.78, and was positively correlated with air and soil temperature, rainfall, and soil moisture with correlation coefficients of 0.48, 0.47, 0.49, and 0.40; the content of TCG1 pathogen in the soil was positively correlated with soil moisture with a correlation coefficient of 0.53. The content of the pathogen in the fruit of the susceptible variety GDS1 at point 1 was not highly correlated with the meteorological factors; the content of TDS1 pathogen in the soil was negatively correlated with soil moisture with a correlation coefficient of 0.51. The content of pathogens in the fruit of the susceptible variety GDS2 at point 2 was positively correlated with total solar radiation, with a high correlation coefficient of 0.70, and positively correlated with air and soil temperature, with correlation coefficients of 0.54 and 0.55, respectively, and negatively correlated with relative humidity and rainfall, with correlation coefficients of 0.51 and 0.46. The content of TDS2 pathogens in the soil was positively correlated with wind speed, rainfall and relative humidity, with correlation coefficients of 0.60, 0.40 and 0.37, respectively, and negatively correlated with total solar radiation and soil moisture, with correlation coefficients of 0.41 and 0.38, respectively. Therefore, the changes in mulberry sclerotinia disease were highly correlated with various meteorological factors, and more samples needed to be collected. It was necessary to use the neural network algorithm to establish a prediction model to further explore the relationship between these factors and achieve accurate prediction of sclerotinia disease.

[0070] 4. Analysis of disease epidemic factors

[0071] A survey of the susceptible fruit mulberry variety DS at the same point found that the incidence rates were 9.06%, 2.75% and 18.59% in 2022, 2023 and 2024, respectively. According to the survey, the initial flowering period to the peak flowering period of the susceptible variety DS in 2022 and 2023 is March 9-March 16. In 2024, due to the damage of freezing rain and blizzards, the fruit mulberry flower buds were damaged and the germination was delayed from March 25 to April 1. Since the initial to peak flowering period is the critical period for pathogen infection, we analyzed the relationship between the occurrence of the disease and the average values ​​of various meteorological factors in this period, and found that the incidence rate was strongly positively correlated with relative humidity and rainfall, with correlation coefficients of 0.99 and 0.98, respectively. Figure 7As shown. Analyzing the meteorological data, we found that the relative humidity from the initial flowering period to the peak flowering period of mulberry in 2022 and 2024 was basically above 50%, with an average relative humidity of 58.70% and 66.92% respectively, while in 2023, there were four days with relative humidity of only 23.82%, 29.47%, 40.41%, and 43.42%, and the average relative humidity was only 49.91%, which was much lower than that in 2022 and 2024. There were 3 and 5 rainfalls from the initial flowering period to the peak flowering period of mulberry in 2022 and 2024, with an average rainfall of 0.10mm and 0.13mm respectively, while the average rainfall in 2022 was only 0.06mm, as shown in Figure 2. Figure 8 shown.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture, characterized in that: The steps include: Step 1: Sample collection Before the fruit mulberry leaves turn green, the buds (fruits), fruits and soil of the disease-susceptible variety Yuezhenda 10 (DS) and the disease-resistant variety Taiwan Changguo mulberry (CG) were collected at the test site. At the same time, spores were captured at the test site using an automatic sampler, and spores on the spore capture strips were collected using a disposable swab. Step 2: Sample DNA extraction Extract the total genomic DNA of the microbial community, use agarose gel electrophoresis to detect the quality of the extracted genomic DNA, and determine the DNA concentration and purity; Step 3: PCR amplification and sequencing library construction Using the above-extracted DNA as a template, PCR amplification is performed, PCR products are recovered using agarose gel, the recovered products are purified using a DNA gel recovery and purification kit, and the recovered products are detected and quantified; the purified PCR products are library-built and sequenced; Step 4: High-throughput sequencing data analysis The double-end original sequencing sequences were quality controlled and spliced: the sequences after quality control splicing were clustered into operational taxonomic units (OTUs) and chimeras were removed. The relative abundance bar graph of the species-level community was analyzed, and the sequencing abundance of three types of mulberry sclerotinia fungi and Soft-coated fungi in the samples was analyzed; Step 5: Analysis of pathogen epidemic patterns The meteorological data collected were used to calculate the relevant meteorological data, and the daily average air temperature, daily average soil temperature, daily rainfall, daily average relative humidity, daily soil moisture content, daily average wind speed, and daily total solar radiation were obtained. The average value of the relevant meteorological data during the sampling period was calculated. The corrplot package in R (v4.41) was used to calculate the abundance values ​​of the three types of mulberry sclerotinia fungus sequence fragments and the Pearson coefficient with the above meteorological factors, and their correlation was analyzed, so as to analyze the factors affecting the epidemic spread of pathogens. Step 6: Analysis of disease epidemic patterns DS mulberry trees were selected at fixed locations for many years to conduct branch surveys, record the total number of fruits and diseased fruits on the branches, and calculate the incidence rate. The biological traits of DS were investigated, and the relationship between meteorological factors and disease incidence during the critical period of pathogen infection was analyzed in combination with high-throughput sequencing results.

2. The method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture according to claim 1, characterized in that: In step 1, the mulberry trees at test site 1 were planted in 2019 with limited root cultivation, with a row spacing of 3m×2m (114°33′E, 30°48′N); the field at test site 2 was planted in 2021, with a row spacing of 3m×2m, and is 500m away from the test base at site 1.

3. The method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture according to claim 1, characterized in that: In the step 1, three DS and three CG plants were selected at test points 1 and 2, three branches were selected from each mulberry tree, and five buds were collected from each branch; at the same time, the soil around each tree was collected, and five points were selected under each tree each time to collect soil samples of the 0-20 cm tillage layer, and debris such as gravel and plant roots were removed. Then, the soil around the three DS and CG plants was mixed and put into a ziplock bag, which was immediately sealed and taken back to the laboratory, and stored in a -80°C refrigerator after being sieved with a 1 mm sieve, and samples were taken every 7 days.

4. The method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture according to claim 1, characterized in that: In step 3, PCR amplification was performed using primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′).

5. The method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture according to claim 1, characterized in that: In the step 3, the PCR reaction system is: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of 5 uM upstream primer, 0.8 μL of 5 μM downstream primer, 0.4 μL of TransStartFastPfu DNA polymerase, 10 ng of template DNA, and the total volume is 20 μL; The amplification program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 27 cycles, then 72°C stable extension for 10 min, and finally storage at 4°C.

6. The method for analyzing the epidemic pattern of mulberry sclerotinia sclerotiorum based on spore capture according to claim 1, characterized in that: In step 6, three DS mulberry trees at a fixed location were selected, three branches of each mulberry tree were randomly surveyed, and each branch was surveyed for a length of 1 m. The total number of fruits and the number of diseased fruits of the 1 m branch were recorded, and the incidence rate was calculated as the number of diseased fruits / total number of fruits×100%.

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