A urease-assisted diagnosis method for lonicera japonica weak disease

Abstract

The application discloses an auxiliary diagnosis method for red bayberry debilitation disease, which comprises collecting healthy red bayberry growth soil and suspected diseased red bayberry growth soil in the same area, and respectively determining urease activity of the soil; and comparing and analyzing urease activity determination results of the healthy red bayberry growth soil and the suspected diseased red bayberry growth soil. If urease activity of the suspected diseased red bayberry growth soil is significantly lower than that of the healthy red bayberry, it is indicated that the suspected diseased red bayberry is likely to have the debilitation disease; if there is no significant difference between urease activity of the suspected diseased red bayberry growth soil and that of the healthy red bayberry growth soil, it is indicated that the suspected diseased red bayberry does not have the debilitation disease. The application can assist in diagnosing the red bayberry debilitation disease, avoids subjective misjudgment of personnel observation diagnosis, and the method is simple, rapid and low in detection cost. In addition, the diagnosis result of the method can provide a reference for further orchard soil treatment and debilitation disease prevention of fruit farmers.

Description

Technical Field

[0001] This invention relates to the field of plant disease diagnosis, specifically a method for assisting in the diagnosis of bayberry weakness disease through soil testing. 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 inflammation, allergies, diabetes, cancer, bacterial infections, and diarrhea. Chinese bayberries are widely cultivated in southern my country, including in Zhejiang, Fujian, Guangdong, Guangxi, Jiangsu, Jiangxi, Yunnan, and Chongqing, with Zhejiang being a major producer.

[0003] The inventors of this invention discovered that *Myrica rubra* suffers from a weakening disease, characterized by: increased fruit yield, smaller fruit size, poor quality, severe leaf drop of mature leaves, and severe tree weakness. This disease primarily affects orchards during their peak production period, with significant outbreaks in Zhejiang, Guangxi, Guangdong, Fujian, and Jiangxi provinces. In severely affected orchards, the mortality rate can reach as high as 80% [Ren Haiying, Zheng Xiliang, Zhang Shuwen, et al., Symptoms of *Myrica rubra* Weakness Disease and Mineral Nutrition Analysis of Diseased Trees, Zhejiang Agricultural Sciences, 2020, 61(10): 2043-2048]. Therefore, *Myrica rubra* weakening disease has a wide incidence and causes significant damage, seriously threatening *Myrica rubra* cultivation and reducing farmers' income. To effectively control *Myrica rubra* weakening disease, it is necessary to diagnose the disease and provide timely and targeted treatment to minimize losses. Summary of the Invention

[0004] The purpose of this invention is to provide an auxiliary diagnostic method for bayberry weakness disease, so as to make timely diagnosis of bayberry weakness disease and facilitate subsequent targeted measures to reduce disease losses.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for the auxiliary diagnosis of bayberry weakness disease, characterized by the following steps: collecting soil samples from healthy bayberry growing areas and soil samples from suspected diseased bayberry growing areas, and measuring the urease activity in the soil samples; comparing and analyzing the urease activity results from the healthy bayberry growing soil and the suspected diseased bayberry growing soil. If the urease activity in the soil from the suspected diseased bayberry growing areas is significantly lower than that in the healthy bayberry growing areas, it indicates that the suspected diseased bayberry is likely to have weakness disease; if there is no significant difference in urease activity between the soil from the suspected diseased bayberry growing areas and the soil from the healthy bayberry growing areas, it indicates that the suspected diseased bayberry does not have weakness disease.

[0007] Furthermore, the pH values ​​of the soil in which the healthy bayberries were grown and the soil in which the suspected diseased bayberries were grown were measured and compared. If the pH value of the soil in which the suspected diseased bayberries were grown was significantly lower than that of the soil in which the healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

[0008] Furthermore, the amount of exchangeable aluminum in the soil from which healthy bayberries were grown and the soil from which suspected diseased bayberries were grown were measured separately, and the two were compared and analyzed. If the amount of exchangeable aluminum in the soil from which suspected diseased bayberries were grown was significantly higher than that in the soil from which healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

[0009] Furthermore, the acid phosphatase activity in the soil from which healthy bayberries were grown and the soil from which suspected diseased bayberries were grown were measured and compared. If the acid phosphatase activity in the soil from which suspected diseased bayberries were grown was significantly lower than that in the soil from which healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

[0010] Preferably, the growing 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, collected using the quartering method and passed through a 0.45mm sieve.

[0011] Furthermore, the region includes Cixi City, Linhai City, Taishun County, Tiantai County, and Wencheng County in Zhejiang Province.

[0012] The advantages of this invention are:

[0013] (1) This invention provides an auxiliary diagnostic method for bayberry weakness disease by measuring urease activity in the soil. Furthermore, measuring soil pH, exchangeable aluminum, and acid phosphatase activity can further determine whether bayberry has contracted weakness disease. This invention provides data support for the diagnosis of bayberry weakness disease, avoiding subjective misjudgments based on human observation.

[0014] (2) The symptoms of waxberry weakness disease include: increased fruit yield, smaller fruit size, poor quality, severe leaf drop of mature leaves, and severe tree weakness. Fruit growers often wait until the waxberries have fruited before determining if the trees are affected by this disease. By then, the fruit is smaller and of lower quality, resulting in poor market returns and significantly impacting the growers' income. This invention allows for diagnosis at any time, especially before the waxberries bear fruit, enabling early treatment and further minimizing losses.

[0015] (3) The diagnostic results of this invention can reflect the soil condition of the orchard and can provide a reference for fruit farmers to further manage the orchard soil and prevent and control debilitating diseases. In addition, the soil of the orchard can be tested regularly using the method of this invention to monitor soil changes. When the urease activity in the soil decreases, the possibility of debilitating disease in bayberries should be considered, and prevention should be carried out as early as possible. Attached Figure Description

[0016] Figure 1 Map showing the selected area of ​​the bayberry orchard

[0017] Figure 2 Comparison of soil enzyme activity results between healthy and diseased bayberry plants. H represents soil from healthy bayberry plants, WD represents soil from diseased bayberry plants, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. Detailed Implementation

[0018] The present invention will be further explained below with reference to the embodiments.

[0019] Example 1: Enzyme Activity and Determination in Soil from Healthy and Weakened Waxberry Plants

[0020] 1. Experimental Materials

[0021] Select bayberry orchards from five major bayberry producing areas in Zhejiang Province: Cixi City (CX), Linhai City (LH), Taishun County (TS), Tiantai County (TT), and Wencheng County (WC). Figure 1 The selected orchards had similar soil and structure, and both contained healthy and weak waxberry trees. The weak waxberry trees exhibited typical symptoms, including a large yield of fruit, small fruit size, poor quality, severe shedding of mature leaves, and severely weakened tree vigor.

[0022] Three bayberry orchards were selected from each of the five regions, with five healthy bayberry trees and five weak or diseased bayberry trees selected from each orchard. Topsoil samples (0-20 cm) were collected from the drip line of the canopy, approximately 1.5 m around the trunk of the bayberry tree, and mixed soil samples were collected using the quartering method.

[0023] 2. Enzyme activity assay methods

[0024] The activities of soil acid phosphatase (ACP) were determined by the disodium phenyl phosphate colorimetric method; the activities of soil urease (URE) were determined by the sodium phenoxide-sodium hypochlorite colorimetric method; the activities of soil catalase (CAT) were determined by the potassium permanganate titration method; and the activities of soil sucrase (S-SC) were determined by the 3,5-dinitrosalicylic acid colorimetric method [Alef K, Nannipieri P. Methods in applied soil microbiology and biochemistry: Academic Press, 1995.].

[0025] 3. Experimental Results and Analysis

[0026] Enzyme activity assay results as follows Figure 2 As shown in the results, the total enzyme activity in the soil where healthy bayberries grow is relatively higher than that in the soil where weakened or diseased bayberries grow. Except for Tiantai County, the acid phosphatase activity in the soil where healthy bayberries grow is significantly higher than that in the soil where weakened or diseased bayberries grow in Cixi City, Linhai City, Taishun County, and Wencheng County. The urease activity in the soil where healthy bayberries grow is significantly higher than that in the soil where weakened or diseased bayberries grow in all five regions. There is no significant difference in soil sucrase activity between the soils where healthy and weakened or diseased bayberries grow in Cixi City, Linhai City, and Taishun County, but the opposite trend is observed in the soils where healthy and weakened bayberries grow in Taishun County and Wencheng County. The catalase activity in the soils where healthy bayberries grow is significantly higher than that in the soils where weakened or diseased bayberries grow in Cixi City and Linhai City; while in the soils where healthy bayberries grow is significantly lower than that in the soils where weakened or diseased bayberries grow in Taishun County and Tiantai County; there is no significant difference between the two in Wencheng County.

[0027] The above experimental results show that: (1) The total activity of various enzymes in the soil where the waxberry grows is reduced. This may be due to the reduced enzyme activity, which leads to a decrease in soil fertility and a reduction in the nutrients required for the growth of the waxberry, thus causing the waxberry to weaken.

[0028] (2) Urease is an enzyme that catalyzes the hydrolysis of urea into ammonia and carbon dioxide, and has a nitrogen-fixing effect, which can promote the effective utilization of nitrogen fertilizer. In addition, under the action of urease, urea decomposes to produce ammonia, which can increase the pH value of the soil, thereby inhibiting soil acidification. The above experimental results show that the urease activity in the soil where weakened bayberries grow is significantly lower than that of healthy bayberries, indicating that urease is an important indicator of bayberry weakness.

[0029] (3) Acid phosphatase degrades organic phosphorus to produce inorganic soluble phosphorus, which is then absorbed and utilized by plant roots. Except for Tiantai County, the acid phosphatase activity in the soil where weakened bayberries grew was significantly reduced in the other four regions, indicating that the decreased acid phosphatase activity makes bayberries more likely to develop weakness disease.

[0030] (4) The trends of soil sucrase and catalase in the soils where weakened and healthy bayberries grow are inconsistent, indicating that the enzyme activity in the soil is affected by a variety of factors. In addition to being related to the weakened disease of bayberries, it may also be related to local climate, microbial species and other factors. The urease activity in the soils where weakened bayberries grow in different production areas is significantly reduced, further illustrating the correlation between urease and the weakened disease of bayberries.

[0031] Example 2: Determination of pH, aluminum, and their content in the soil from which healthy and weakened / disease-stricken bayberries grow.

[0032] 1. Experimental Materials

[0033] Same as Example 1

[0034] 2. Measurement Method

[0035] (1) pH value

[0036] A suspension was prepared with a soil:water ratio of 1:2.5 (w / v) and the pH was measured using a pH meter (PB-21 pH meter, Sartorius, Germany).

[0037] (2) Aluminum

[0038] Extraction of aluminum in different chemical forms using different solvents [Wu Chunhua, Yu Wei, Yin Jiayuan, et al., Dissolution and speciation of active aluminum in sludge, Environmental Chemistry, 2001, 20: 262-264]. Aluminum was extracted sequentially with 1M potassium chloride solution, 1M ammonium acetate solution, 1M hydrochloric acid, and 0.5M sodium hydroxide solution, yielding exchangeable aluminum (ExAl) and monomeric hydroxyl aluminum (e.g., Al(OH)). 2+ and Al(OH)2 + The aluminum compounds were denoted as HyAl, colloidal aluminum (CoAl), and humic-acid aluminum (HaAl). Each extraction involved centrifugation at 3500 rpm for 30 min, followed by filtering through a 0.45 μm filter to separate the solid and liquid phases, yielding the extract. Aluminum in each extract was determined using the chromium azurol S (CAS) spectrophotometric method [Pakalns P. Spectrophotometric determination of aluminum with chrome azurol S. Analytica Chimica Acta 1965, 32:57-63.].

[0039] 3. Experimental Results and Analysis

[0040] The pH results of the soil for waxberry growth are shown in Table 1 below:

[0041] Table 1. Results of pH value determination in soil for waxberry growth.

[0042]

[0043] Note: ns indicates no significant difference.

[0044] The results of aluminum determination in different chemical forms in the soil where bayberry grows are shown in Table 2 below:

[0045] Table 2. Results of aluminum determination in different forms in the soil where Yangmei (Myrica rubra) grows.

[0046]

[0047] 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.

[0048] The experimental results show that all soils had acidic pH values, all below 5.0. Except for Linhai City, the pH values ​​of the soils where weakened and diseased bayberries grew in the other four regions were significantly lower than those of healthy bayberries. The concentration order of the four forms of aluminum in the soil was: HaAl > CoAl > ExAl > HyAl. Except for Linhai City and Tiantai County, the ExAl levels in the soils where weakened and diseased bayberries grew in the other three regions were significantly higher than those of healthy bayberries.

[0049] The results of this experiment show that: (1) the soil in which bayberries grow is acidified to a certain extent. Chai Chunyan et al. reported that in the past 30 years, the soil pH of bayberry orchards in Zhejiang Province has decreased by 1.31 units, and soil acidification has increasingly affected the yield and quality of bayberries. [Chai Chunyan, Song Xuzhong, Xu Shaoqing, et al. Causes and improvement measures of soil acidification in bayberry orchards. Modern Horticulture, 2015, 14: 217-219]. The soil pH of the bayberry growing in the diseased bayberry is relatively lower than that of the healthy bayberry growing soil, which further illustrates that soil pH is closely related to the occurrence of bayberry disease. The lower the pH, the greater the probability of bayberries getting the disease.

[0050] (2) Aluminum in soil mainly exists in two forms: HaAl and CoAl. HaAl is formed by aluminum combining with insoluble organic matter in the soil, while CoAl is formed by aluminum adsorbed onto soil minerals. Both forms of aluminum are not easily dissolved and have little toxicity to plants. ExAl is a soluble inorganic monomer of aluminum and has a greater toxicity to plants. The concentration of ExAl in the soil where *Myrica rubra* var. *mongolica* grows is higher, indicating that soil ExAl is related to the occurrence of *Myrica rubra* var. *mongolica* var. *mongolica* var. *mongolica* var. *mongolica*. In addition, the lower the soil pH, the easier it is for ExAl to form in the soil, which is consistent with the pH results.

[0051] (3) Combined with the urease experiment results, it can be seen that urease can decompose urea to produce ammonia, which increases the soil pH. The soil of the weakened bayberry plant showed lower urease activity, lower pH, and higher ExAl concentration, consistent with these findings. This result indicates that the occurrence of bayberry weakening disease may be due to reduced urease activity in the soil, decreased ammonia production, and inability to effectively neutralize soil acid, leading to a decrease in soil pH, which in turn increases ExAl, poisoning the bayberry roots and causing weakness. Therefore, detecting urease activity in the soil can help predict the likelihood of weakening disease and allow for timely prevention, reducing the risk of disease.

[0052] Example 3: Analysis of various indicators and comparative analysis of soil samples from suspected diseased bayberry plants.

[0053] In December, healthy waxberry trees with good growth and suspected diseased waxberry trees with fallen mature leaves were selected from the same region as in Example 1. Topsoil samples (0-20 cm) were collected from the drip line of the canopy, approximately 1.5 m around the waxberry tree trunk, and the mixed soil samples were collected using the quartering method. Urease activity, acid phosphatase activity, pH value, and bound aluminum in the soil were measured according to the experimental methods in Examples 1 and 2. The growth status of the waxberry fruits was observed and recorded when the waxberries ripened (June-July). The experimental results are shown in Table 3 below (one healthy waxberry tree and two suspected diseased waxberry trees were selected from each region).

[0054] Table 3. Results of various indicators in healthy and suspected diseased bayberries.

[0055]

[0056]

[0057] Remark: * This indicates a significant difference between those suspected of having the disease and those who are healthy (p<0.05). ** The above indicates a highly significant difference between those suspected of having the disease and those who are healthy (p<0.01); ns This indicates that there is no significant difference between those suspected of having the disease and those who are healthy.

[0058] The experimental results show that, compared with healthy bayberries, those suspected of being diseased, exhibiting a significant decrease in soil urease activity, produced large but small fruits, indicating weakness disease. Conversely, those with no significant decrease in soil urease activity produced normal fruits, not showing signs of weakness disease. These results demonstrate that using soil urease activity measurement as an auxiliary diagnostic tool for bayberry weakness disease is feasible. Furthermore, this method allows for timely diagnosis of suspected diseased plants before fruiting, enabling early detection and treatment, and reducing losses.

[0059] In addition, in suspected diseased plants, the fruit is abundant but small. In confirmed cases of weakened waxberry disease, compared to healthy waxberry plants, the soil in these plants often shows significantly lower acid phosphatase activity, significantly higher exchangeable aluminum, and significantly lower pH. Therefore, measuring soil acid phosphatase activity, exchangeable aluminum, and pH before waxberry fruiting can further aid in determining whether waxberries are suffering from weakened disease.

Claims

1. An auxiliary diagnostic method for myrica rubra debilitaria, characterized in that, The method includes: collecting soil samples from healthy bayberry growing areas and soil samples from suspected diseased bayberry growing areas, and measuring the urease activity of the soil samples respectively; comparing and analyzing the urease activity measurement results of the healthy bayberry growing soil and the suspected diseased bayberry growing soil; if the urease activity in the soil samples from suspected diseased bayberries is significantly lower than that in healthy bayberries, it indicates that the suspected diseased bayberries are likely to have senility disease; if there is no significant difference between the urease activity in the soil samples from suspected diseased bayberries and that in the soil samples from healthy bayberries, it indicates that the suspected diseased bayberries have not developed senility disease.

2. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The pH values ​​of the soil in which healthy bayberries were grown and the soil in which suspected diseased bayberries were grown were measured and compared. If the pH value of the soil in which suspected diseased bayberries were grown was significantly lower than that in the soil in which healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

3. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The amount of exchangeable aluminum in the soil from which healthy bayberries were grown and the soil from which suspected diseased bayberries were grown were measured and compared. If the amount of exchangeable aluminum in the soil from which suspected diseased bayberries were grown was significantly higher than that in the soil from which healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

4. The auxiliary diagnostic method for myrica fulminans disease according to claim 1, characterized in that, The acid phosphatase activity in the soil from which healthy bayberries were grown and the soil from which suspected diseased bayberries were grown were measured and compared. If the acid phosphatase activity in the soil from which suspected diseased bayberries were grown was significantly lower than that in the soil from which healthy bayberries were grown, it indicated that the suspected diseased bayberries were more likely to have senility disease.

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

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

7. The use of soil urease activity in the auxiliary diagnosis of bayberry weakness disease, characterized in that, The auxiliary diagnostic method includes: collecting soil samples from healthy bayberry growing areas and soil samples from suspected diseased bayberry growing areas, and measuring the urease activity in the soil samples respectively; comparing and analyzing the urease activity measurement results of the healthy bayberry growing soil and the suspected diseased bayberry growing soil; if the urease activity in the soil sample from suspected diseased bayberry growing areas is significantly lower than that in healthy bayberry growing areas, it indicates that the suspected diseased bayberry is likely to have debilitating disease; if there is no significant difference between the urease activity in the soil sample from suspected diseased bayberry growing areas and that in the soil sample from healthy bayberry growing areas, it indicates that the suspected diseased bayberry is not diseased.

8. The use of soil urease activity according to claim 7 in the auxiliary diagnosis of bayberry weakness disease, characterized in that, The region includes Cixi City, Linhai City, Taishun County, Tiantai County, and Wencheng County in Zhejiang Province.

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