A fluorescent quantitative PCR assisted diagnosis method for blueberry weak disease

Abstract

The application discloses an auxiliary diagnosis method for red bayberry weakening disease, which comprises collecting growth soil of healthy red bayberry and suspected diseased red bayberry, respectively determining relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil, and if the relative abundance of Acidothermus bacteria in the growth soil of the suspected diseased red bayberry is significantly reduced, and the relative abundance of Cladophialophora fungi is significantly increased, compared with the growth soil of the healthy red bayberry, it is indicated that the suspected diseased red bayberry has a high possibility of weakening disease, and if the relative abundance of the amount of Acidothermus bacteria and Cladophialophora fungi in the growth soil of the suspected diseased red bayberry and the growth soil of the healthy red bayberry has no significant difference, it is indicated that the suspected diseased red bayberry does not have weakening disease. The application provides a new rapid molecular auxiliary diagnosis method for red bayberry weakening disease, and the method can be carried out at any time, and can effectively reduce the loss of fruit farmers.

Description

Technical Field

[0001] This invention relates to the field of plant disease diagnosis, specifically a method for auxiliary diagnosis of bayberry weakness disease by quantitative analysis of soil microorganisms using fluorescent PCR. Background Technology

[0002] The Chinese bayberry (Myrica rubra Sieb. et Zucc.) is an important specialty fruit tree in southern China. Its fruit has a balanced sweet and sour taste and a unique flavor, enjoying a high reputation both domestically and internationally. Furthermore, the Chinese bayberry is also an important medicinal plant; extracts from its fruit have adjunctive therapeutic effects on diseases such as inflammation, allergies, diabetes, cancer, bacterial infections, and diarrhea. The Chinese bayberry is widely cultivated in southern my country, generating significant economic benefits.

[0003] The inventors of this invention discovered a weakening disease in the waxberry tree, characterized by increased fruit yield, smaller fruit size, poor quality, severe leaf drop of mature leaves, and overall tree weakness. The disease is graded according to a disease index as follows: Grade 0: Dense foliage and healthy tree; Grade 1: 0 < leaf drop ≤ 10% of total leaf area; Grade 3: 10% < leaf drop ≤ 25% of total leaf area; Grade 5: 25% < leaf drop ≤ 50% of total leaf area; Grade 7: 50% < leaf drop ≤ 75% of total leaf area; Grade 9: 75% < leaf drop ≤ 100% of total leaf area. Investigations revealed that this weakening disease is characterized by its severe impact and wide geographical distribution. It primarily occurs in orchards during peak production periods, with significant outbreaks in Zhejiang, Guangxi, Guangdong, Fujian, and Jiangxi provinces. In orchards with severe disease outbreaks, the mortality rate can even reach 80% [Ren Haiying, Zheng Xiliang, Zhang Shuwen, et al. Symptoms of Yangmei Weakness Disease and Mineral Nutrition Analysis of Diseased Trees, Zhejiang Agricultural Sciences, 2020, 61(10): 2043-2048].

[0004] In summary, the disease of weakness in bayberries has a wide range of incidence and causes significant damage, seriously threatening the cultivation of bayberries and reducing the income of fruit growers. To effectively control the disease, it is necessary to diagnose it and provide timely, targeted treatment to minimize losses. Currently, the diagnosis of bayberry weakness relies solely on the subjective observation of fruit growers, and there is no scientific diagnostic method. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide an auxiliary diagnostic method for myrica rubra fasciitis.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] An auxiliary diagnostic method for bayberry debilitation disease includes: collecting soil samples from healthy bayberries and suspected diseased bayberries in the same region, and determining the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in the soil; comparing the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in the soil samples from suspected diseased bayberries with that from healthy bayberries; if the relative abundance of *Acidothermus* bacteria is significantly lower and the relative abundance of *Cladophialophora* fungi is significantly higher in the soil samples from suspected diseased bayberries compared to the soil samples from healthy bayberries, then the suspected diseased bayberries are likely to have debilitation disease; if there is no significant difference in the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in the soil samples from suspected diseased bayberries, then the suspected diseased bayberries are not diseased.

[0008] Furthermore, the procedure for determining the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil includes: extracting genomic DNA from the soil sample using a DNA extraction kit, and using the genomic DNA as a template, selecting specific primers for quantitative analysis by fluorescent PCR.

[0009] Preferably, the specific primers include: an upstream primer 5'-CTGCCCTTGACTCTGGGATA-3' (Tm=60.21℃) and a downstream primer 5'-GTCGCCTTGGTAAGCCATTA-3' (Tm=60.10℃) for Acidothermus bacteria, and an upstream primer 5'-GGTTGTCACCAATCCTCTGG-3' (Tm=60.36℃) and a downstream primer 5'-ATTTCGCTGCGTTCTTCATC-3' (Tm=60.36℃) for Cladophialophora fungi. The PCR product length of Acidothermus bacteria is 154, and the PCR product length of Cladophialophora fungi is 158.

[0010] Furthermore, the soil is the top 0-20cm layer of soil collected from the drip line of the canopy, about 1.5m around the trunk of the bayberry tree. It is collected using the quartering method, passed through a 0.45mm sieve, and stored at low temperature.

[0011] Preferably, the soil from the production of bayberries is collected regularly, and the amount of Acidothermus bacteria and Cladophialophora fungi in the soil is measured. By monitoring the changes in Acidothermus bacteria and Cladophialophora fungi, the possibility of bayberries being susceptible to debilitating disease can be monitored.

[0012] Furthermore, the region includes Cixi City, Linhai City, Taishun County, Tiantai County, and Wencheng County in Zhejiang Province.

[0013] The advantages of this invention are as follows: (1) This invention provides an auxiliary diagnostic method for bayberry weakness disease by measuring the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil, which can quickly and effectively obtain diagnostic results and provide a new molecular diagnostic means for bayberry weakness disease. (2) By regularly detecting and monitoring the relative abundance of Acidothermus bacteria and Cladophialophora fungi in bayberry orchards using the method described above, the possibility of bayberry weakness disease can be detected in time, and prevention and control can be carried out as early as possible. (3) Bayberry weakness disease is characterized by: increased fruit yield, smaller fruit size, poor quality, severe shedding of mature leaves, and severe tree weakness. When observing and diagnosing, fruit farmers often wait until the bayberries have fruited before they can determine whether the fruit trees have weakness disease. At this time, the fruit size is small, the quality is poor, and good profits cannot be obtained, which has seriously affected the income of fruit farmers. This invention can be used for diagnosis at any time, especially before the bayberries have fruited, so as to carry out treatment as early as possible and further reduce losses. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments.

[0015] Example 1: Comparative Study of Microorganisms in the Soil Where Healthy and Weak / Disease-Induced Waxberry Trees Grow

[0016] (1) Experimental materials

[0017] We selected bayberry orchards from five major bayberry-producing areas in Zhejiang Province: Haining City, Linhai City, Taishun County, Tiantai County, and Wencheng County. All selected orchards contained both healthy and weakened / disease-prone bayberries.

[0018] Choose healthy bayberry trees and weak or diseased bayberry trees whose crown size and leaf loss account for 25% to 50% of the total leaves on the tree (disease index level 5).

[0019] (2) Soil sample collection

[0020] Topsoil samples (0-20cm) were collected from the drip line of the canopy, approximately 1.5m around the trunk of the bayberry tree. Mixed soil samples were collected using the quartering method, passed through a 0.45mm sieve, and stored in a refrigerator at -80℃.

[0021] (3) Soil genome sequencing

[0022] Genome sequencing was performed by Shanghai DNeasy Biomedical Technology Co., Ltd. Genomic DNA was extracted from soil samples using DNA extraction kits (DNeasy PowerSoil Kit, QIAamp 96 PowerFecal QIAcube HT kit). Using diluted genomic DNA as a template, PCR was performed using Tks Gflex DNA Polymerase high-fidelity enzyme (Takara, Dalian). Barcode-specific primers 343F: 5'-TACGGRAGGCAGCAG-3' and 798R: 5'-AGGGTATCTAATCCT-3' were used to amplify the 16S rRNA V3-V4 region corresponding to bacterial diversity. ITS1F-5'-CTTGGTCATTTAGAGGAAGTAA-3' and ITS2-5'-GCTGCGTTCTTCATCGATGC-3' were selected to amplify the ITS1 and ITS2 regions corresponding to fungal ITS diversity. After quantification using the Qubit dsDNA Assay Kit, PCR products were pooled in equal volumes and sequenced using the Illumina MiSeq PE300 platform. Microbial diversity was analyzed using Illumina MiSeq sequencing, and OTUs were classified using Vsearch software at 97% similarity. Representative sequences were annotated against databases using the RDP Classifier NaiveBayesian.

[0023] (4) Experimental results

[0024] Illumina MiSeq high-throughput sequencing results showed that the bacterial library yielded 20,020–38,808 valid sequences, with an average sequence length ranging from 422.57 to 430.85 bp. At a 97% similarity level, the number of OTUs in each sample ranged from 1,482 to 2,727. The fungal library yielded 21,914–134,217 valid sequences, with an average sequence length ranging from 258 to 319 bp. At a 97% similarity level, the number of OTUs in each sample ranged from 608 to 1,168. The number of bacterial OTUs shared among the different treatments was 279. The average number of OTUs in healthy trees (H) was 1,685, while the average number of OTUs in weakened and diseased trees (D) was 1,575. The number of bacterial OTUs in the rhizosphere soil of weakened and diseased trees was 9.78% lower than that in the healthy tree group.

[0025] The relative abundance of 10 bacteria with relatively high levels of abundance in the soil where bayberry grows in various regions is shown in Table 1 below. The relative abundance of 10 fungi with relatively high levels of abundance in the soil where bayberry grows in various regions is shown in Table 2 below.

[0026] Table 1. Comparison of the results of determination of 10 bacteria (genus level) with relatively high abundance in the soil of bayberry growing regions.

[0027]

[0028] Remark: * This indicates a significant difference between frail patients and healthy patients, p<0.05; ** The presence of frailty between healthy and unhealthy individuals was highly significant (p < 0.01). ns This indicates that there is no significant difference between those with frailty and those who are healthy.

[0029] Table 2. Comparison of the results of determination of 10 fungi (genus level) with relatively high abundance in the soil of bayberry growing in different regions.

[0030]

[0031] Remark: * This indicates a significant difference between frail patients and healthy patients, p<0.05; ** The presence of frailty between healthy and unhealthy individuals was highly significant (p < 0.01). ns This indicates that there is no significant difference between those with frailty and those who are healthy.

[0032] Table 1 shows that among the 10 bacterial species, the relative abundance of *Acidothermus* bacteria in the soil where *Myrica rubra* grew in weakened varieties was significantly lower than that in healthy varieties in all regions, indicating a close relationship between *Acidothermus* bacteria in the soil and *Myrica rubra* weakening disease. The trends of the other 9 bacterial species varied across regions, indicating significant differences in soil bacteria and diverse influencing factors. However, the consistent trend of *Acidothermus* bacteria across regions further confirms the close relationship between *Acidothermus* bacteria and *Myrica rubra* weakening disease.

[0033] Table 2 shows that among the 10 fungal species, the abundance of *Cladophialophora* fungi in the soil of weakened *Myrica rubra* in all regions was significantly higher than that in healthy *Myrica rubra*, indicating that changes in *Acidothermus* bacteria and *Cladophialophora* fungi in the soil are closely related to *Myrica rubra* weakness. The trends of the other 9 fungal species varied across regions, indicating significant differences in soil fungi and diverse factors influencing fungi. However, the consistent trend of *Cladophialophora* fungi across regions further demonstrates the close relationship between *Cladophialophora* fungi and *Myrica rubra* weakness.

[0034] Example 2: Determination and comparative analysis of the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil where bayberries grow.

[0035] 1. Using the specific primers for Acidothermus bacteria and Cladophialophora fungi as described in Table 3, perform fluorescence PCR quantitative analysis on the genomic DNA in the soil from which healthy and weak / disease-infected bayberry trees grew as described in Example 1, and determine the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil.

[0036] Table 3. Specific primers for Acidothermus bacteria and Cladophialophora fungi

[0037]

[0038] The experimental results showed that the relative abundance of *Acidothermus* bacteria in the soil where *Myrica rubra* grew in various regions was significantly lower than that in healthy *Myrica rubra* (p<0.01), approximately 83% of that in healthy *Myrica rubra*, while the relative abundance of *Cladophialophora* fungi was significantly higher than that in healthy *Myrica rubra* (p<0.001), approximately 175% of that in healthy *Myrica rubra*. This result further confirms that *Acidothermus* bacteria and *Cladophialophora* fungi in the soil are closely related to *Myrica rubra* weakness disease, and the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in the soil can be used as diagnostic indicators for *Myrica rubra* weakness disease.

[0039] 2. In December, healthy bayberries and suspected diseased bayberries exhibiting signs of leaf drop and weakened growth were selected from the aforementioned orchard. Soil samples were taken according to Example 1, and genomic DNA was extracted from the soil samples using a DNA extraction kit (DNeasy PowerSoil Kit, QIAamp 96 PowerFecal QIAcube HT kit). Quantitative analysis was performed using fluorescent PCR with specific primers for *Acidothermus* bacteria and *Cladophialophora* fungi. The fruit condition of the healthy and suspected diseased bayberries was observed and recorded the following year when the bayberries ripened (June-July).

[0040] The experimental results showed that, compared with the soil in which healthy bayberries grew, the suspected diseased bayberries with a significantly reduced relative abundance of Acidothermus bacteria and a significantly increased relative abundance of Cladophialophora fungi produced large fruits with small fruit size, exhibiting symptoms of weakness disease. In contrast, the fruits of suspected diseased bayberries with no significant reduction in the relative abundance of Acidothermus bacteria or no significant increase in the relative abundance of Cladophialophora fungi were normal and did not show symptoms of weakness disease.

[0041] The above results confirm that by measuring the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil where suspected diseased bayberries grow and comparing it with that in the soil where healthy bayberries grow, it is possible to diagnose whether bayberries are suffering from debilitating disease. In addition, this diagnostic method can be performed at any time, especially before the bayberries bear fruit, so treatment measures can be taken before the fruit trees bear fruit to further reduce losses.

Claims

1. An auxiliary diagnostic method for myrica rubra debilitaria, characterized in that, The method includes: collecting soil samples from healthy and suspected diseased bayberries in the same region, and determining the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil; comparing the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil of suspected diseased bayberries with that of healthy bayberries; if the relative abundance of Acidothermus bacteria is significantly lower and the relative abundance of Cladophialophora fungi is significantly higher in the soil of suspected diseased bayberries compared to the soil of healthy bayberries, it indicates that the suspected diseased bayberries are likely to have senility disease; if there is no significant difference in the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil of suspected diseased bayberries, it indicates that the suspected diseased bayberries have not had senility disease.

2. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The steps for determining the relative abundance include: extracting genomic DNA from the soil using a DNA extraction kit, and using the genomic DNA as a template, selecting specific primers for quantitative analysis by fluorescent PCR.

3. The auxiliary diagnostic method for myrica fulminans disease according to claim 2, characterized in that, The specific primers include: upstream primer 5'-CTGCCCTTGACTCTGGGATA-3' and downstream primer 5'-GTCGCCTTGGTAAGCCATTA-3' for Acidothermus bacteria, and upstream primer 5'-GGTTGTCACCAATCCTCTGG-3' and downstream primer 5'-ATTTCGCTGCGTTCTTCATC-3' for Cladophialophora fungi.

4. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The soil in question was the top 0-20cm layer collected from the drip line of the canopy, which is 1.5m around the trunk of the bayberry tree.

5. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The region includes Haining City, Linhai City, Taishun County, Tiantai County, and Wencheng County in Zhejiang Province.

6. The use of the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in soil in the diagnosis of *Myrica rubra* debilitating disease, characterized in that... The diagnosis includes: collecting soil samples from healthy and suspected diseased bayberries grown in the same area, and measuring the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil; comparing the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil from suspected diseased bayberries with that of healthy bayberries; if the relative abundance of Acidothermus bacteria is significantly lower and the relative abundance of Cladophialophora fungi is significantly higher in the soil from suspected diseased bayberries compared to the soil from healthy bayberries, it indicates that the suspected diseased bayberries are likely to have debilitating disease; if there is no significant difference in the relative abundance of Acidothermus bacteria and Cladophialophora fungi in the soil from suspected diseased bayberries, it indicates that the suspected diseased bayberries are not diseased.

7. The use of the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in soil in the diagnosis of *Myrica rubra* debilitating disease, as described in claim 6, is characterized in that... The steps for determining the relative abundance include: extracting genomic DNA from the soil using a DNA extraction kit, and using the genomic DNA as a template, selecting specific primers for quantitative analysis by fluorescent PCR.

8. The use of the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in soil in the diagnosis of *Myrica rubra* debilitating disease, as described in claim 7, is characterized in that... The specific primers include: upstream primer 5'-CTGCCCTTGACTCTGGGATA-3' and downstream primer 5'-GTCGCCTTGGTAAGCCATTA-3' for Acidothermus bacteria, and upstream primer 5'-GGTTGTCACCAATCCTCTGG-3' and downstream primer 5'-ATTTCGCTGCGTTCTTCATC-3' for Cladophialophora fungi.

9. The use of the relative abundance of *Acidothermus* bacteria and *Cladophialophora* fungi in soil in the diagnosis of *Myrica rubra* debilitating disease, as described in claim 6, is characterized in that... The region includes Haining City, Linhai City, Taishun County, Tiantai County, and Wencheng County in Zhejiang Province.

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