A method for auxiliary diagnosis of weak disease of Myrica rubra by measuring the contents of paratose and cytidine in root soil

Abstract

The present invention discloses a method for auxiliary diagnosis of the decline disease of Myrica rubra by measuring the contents of palatinose and cytidine in the root soil. The method includes: collecting the root soil of healthy Myrica rubra and suspected diseased Myrica rubra in the same area; measuring the contents of palatinose and cytidine in the root soil of healthy Myrica rubra and suspected diseased Myrica rubra and conducting comparative analysis; if the contents of palatinose and cytidine in the root soil of suspected diseased Myrica rubra are both significantly increased compared with those in the root soil of healthy Myrica rubra, it indicates that the suspected diseased Myrica rubra has a high possibility of suffering from the decline disease, and if there is no significant difference in the contents of palatinose and cytidine in the root soil of suspected diseased Myrica rubra, it indicates that the suspected diseased Myrica rubra does not suffer from the decline disease. The present invention provides a new auxiliary diagnosis method for the decline disease of Myrica rubra, and the method can be carried out at any time, enabling early diagnosis and early treatment, and reducing the losses of fruit farmers.

Description

Technical Field

[0001] The present invention relates to the field of plant disease diagnosis, and specifically to a method for assisting in diagnosing the decline disease of Myrica rubra by measuring the contents of palatinose and cytidine in the root soil. Background Art

[0002] Myrica rubra Sieb. Et Zucc is an important precious fruit tree in southern China. Its fruits are moderately sweet and sour, with a unique flavor, and enjoy a high reputation at home and abroad. Moreover, Myrica rubra is also an important medicinal plant. The extracts of Myrica rubra fruits have adjuvant therapeutic effects on diseases such as inflammation, allergy, diabetes, cancer, bacterial infection, and diarrhea. Myrica rubra is widely planted in southern China and has good economic benefits.

[0003] The inventors of the present invention found that in recent years, there has been a decline disease in Myrica rubra, and the symptoms are as follows: the fruit-bearing amount of Myrica rubra trees increases, the fruits become smaller, the quality is inferior, the old leaves fall off seriously, and the tree vigor is severely weakened. According to the disease severity level, it is classified as follows: level 0, the whole tree has dense leaves and the tree vigor is healthy; level 1, 0 < the proportion of the leaf fall amount in the total leaf amount of the whole tree ≤ 10%; level 3, 10% < the proportion of the leaf fall amount in the total leaf amount of the whole tree ≤ 25%; level 5, 25% < the proportion of the leaf fall amount in the total leaf amount of the whole tree ≤ 50%; level 7, 50% < the proportion of the leaf fall amount in the total leaf amount of the whole tree ≤ 75%; level 9, 75% < the proportion of the leaf fall amount in the total leaf amount of the whole tree ≤ 100%. Through investigation, it was found that: the decline disease of Myrica rubra is characterized by severe harm and wide geographical distribution. The disease mainly occurs in orchards during the peak production period, and there are large areas of occurrence in Zhejiang, Guangxi, Guangdong, Fujian, Jiangxi and other places. In severely affected orchards, the mortality rate can even reach 80% [Ren Haiying, Zheng Xiliang, Zhang Shuwen, et al., Analysis of the symptoms and mineral nutrition of Myrica rubra decline disease, Journal of Zhejiang Agricultural Sciences, 2020, 61(10): 2043 - 2048].

[0004] In summary, the decline disease has a wide incidence range and great harm, seriously threatening the planting of Myrica rubra and reducing the income of fruit farmers. In order to effectively prevent and control the decline disease of Myrica rubra, it is necessary to diagnose the decline disease of Myrica rubra in order to carry out targeted treatment in a timely manner and reduce losses. At present, the diagnosis of the decline disease of Myrica rubra is only subjectively judged by fruit farmers, and there is no scientific diagnosis method. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a method for assisting in diagnosing the decline disease of Myrica rubra by measuring the contents of palatinose and cytidine in the root soil.

[0006] The purpose of the present invention is solved by the following technical solutions:

[0007] A method for auxiliary diagnosis of bayberry decline disease by measuring the contents of palatinose and cytidine in root-zone soil, the method comprising: collecting root-zone soil of healthy bayberry and suspected diseased bayberry in the same area; measuring the contents of palatinose and cytidine in the root-zone soil of healthy bayberry and suspected diseased bayberry and performing comparative analysis; if the contents of palatinose and cytidine in the root-zone soil of suspected diseased bayberry are both significantly increased compared with those in the root-zone soil of healthy bayberry, it indicates that the suspected diseased bayberry has a high probability of suffering from decline disease, and if there is no significant difference in the contents of palatinose and cytidine in the root-zone soil of suspected diseased bayberry, it indicates that the suspected diseased bayberry does not suffer from decline disease.

[0008] The specific steps for measuring the contents of palatinose and cytidine in root-zone soil include:

[0009] (1) Take the root-zone soil, add methanol-water (v / v = 1:1) according to a solid-liquid ratio of 1:1 g / ml, add L-2-chlorophenylalanine as an internal standard, grind for 2 min at 60 Hz, centrifuge, take the supernatant, freeze-dry to obtain a powder for standby.

[0010] (2) Take the powder, add an excessive amount (4V) of methoxyamine hydrochloride pyridine solution, shake at 37 °C for 90 min, dry with nitrogen, add 4V of BSTFA solution containing 1% TMCS and V of n-hexane solution, react at 70 °C for 60 min to obtain a silylated sample solution.

[0011] (3) Detect the silylated sample solution by GC-MS.

[0012] The root-zone soil is the surface soil of 0-20 cm collected around the drip line of the tree crown.

[0013] The area includes Haining City, Zhejiang Province.

[0014] The advantages of the present invention are: (1) The present invention discloses a method for auxiliary diagnosis of bayberry decline disease by measuring the contents of palatinose and cytidine in root-zone soil, providing a new diagnostic means for bayberry decline disease.

[0015] (2) The manifestations of bayberry decline disease are: the fruit yield of bayberry trees increases, the fruits become smaller, the quality is poor, the old leaves fall off seriously, and the tree vigor is severely weakened. When fruit farmers observe and diagnose, they usually wait until the leaves fall off and the bayberries bear fruit before they can determine whether the fruit trees are suffering from decline disease. At this time, the fruits are smaller and of poor quality, and they cannot obtain good benefits, which has seriously affected the income of fruit farmers. The present invention can be diagnosed at any time, especially before the bayberries bear fruit, so as to carry out treatment as early as possible and further reduce losses.

[0016] (3) By the method, the contents of palatinose and cytidine in the root-zone soil of bayberry can be regularly detected, the possibility of bayberry suffering from decline disease can be found in time, and prevention and control can be carried out as early as possible. Description of the Drawings

[0017] Figure 1 Total Ion Chromatogram (TIC) of the soil of healthy Myrica rubra root system measured by GC - MSD.

[0018] Figure 2 Total Ion Chromatogram (TIC) of the soil of Myrica rubra with declining disease root system measured by GC - MSD.

[0019] Figure 3 Heat map of metabolites and relative contents in the soil of healthy Myrica rubra and Myrica rubra with declining disease root systems, where H refers to healthy Myrica rubra and W refers to Myrica rubra with declining disease. Detailed implementation mode

[0020] The present invention will be further described below in conjunction with embodiments.

[0021] Example 1 Analysis of metabolites and comparison in the soil of healthy Myrica rubra and Myrica rubra with declining disease root systems

[0022] 1 Experimental materials

[0023] Select a Myrica rubra orchard in Haining City, Zhejiang Province (latitude 30°32'; longitude 120°42') as the test orchard. The environmental conditions of the test orchard are typical gentle slope mountains, about 50 m above sea level. About 65% of the fruit trees in the orchard have declining disease, with disease indices ranging from 1 to 9, and the test orchard is managed conventionally.

[0024] Select 6 Myrica rubra trees with a leaf shedding amount accounting for 25% - 50% of the total leaves of the whole tree (disease index is 5) in the test orchard as the test trees for declining disease.

[0025] Select 6 Myrica rubra trees with dense leaves and healthy tree vigor in the test orchard as the healthy test trees.

[0026] Collect surface soil samples of 0 - 20 cm around the drip line of the test tree crown, collect mixed soil samples using the quartering method, and pass through a 0.45 mm sieve. 6 soil samples are collected in parallel for each tree.

[0027] 2 Sample preparation

[0028] (1) Pretreatment: Weigh 1 g of soil sample, add 20 μl of 0.3 mg / ml L-2-chlorophenylalanine (Shanghai Hengchuang Biotechnology Co., Ltd.) methanol solution as internal standard, add 1 ml of methanol-water (v / v = 1:1) and 2 small steel balls. Pre-cool at -20 °C for 2 min and grind at a frequency of 60 Hz for 2 min. Transfer the well-ground sample to a 15-ml centrifuge tube. During the transfer process, rinse the sample container with 1 ml of methanol-water (v / v = 1:1) and repeat twice. Transfer the rinsing solution to the same 15-ml centrifuge tube. Centrifuge the total 3 ml of sample solution at 4 °C and 7700 rpm for 10 min. Take 2.5 ml of the supernatant to a 5-ml centrifuge tube, freeze-dry to obtain powder. Add 400 μl of methanol-water (v / v = 1:1), vortex for 60 s and then sonicate for 30 s for reconstitution. Transfer the reconstituted solution to a 1.5-ml centrifuge tube, centrifuge at 4 °C and 12,000 rpm for 10 min, and take 300 μl of the supernatant to a derivatization tube, freeze-dry (mix the supernatants of samples with the same volume to make the total volume 300 μl, and its subsequent treatment is the same as that of each sample, serving as a control sample).

[0029] (2) Derivatization: Add 80 μl of 15 mg / ml methoxyamine hydrochloride (CNW Technologies GmbH) pyridine solution to the derivatization tube, vortex for 2 min, and shake at 37 °C for 90 min to fully carry out the oximation reaction. After drying with nitrogen, add 80 μl of BSTFA (containing 1% TMCs, CNW Technologies GmbH) and 20 μl of n-hexane. After vortexing for 2 min, react at 70 °C for 60 min.

[0030] 3 GC-MS Chromatographic Conditions

[0031] Instrument model: 7890B-5977A GC-MSD (Agilent, USA).

[0032] Chromatographic column: DB-5MS capillary column (30 m × 0.25 mm, 0.25 μm; Agilent, USA).

[0033] Carrier gas: High-purity helium (purity not less than 99.999%).

[0034] Carrier gas flow rate: 1 ml / min.

[0035] Injection port temperature: 260 °C.

[0036] Injection volume: 1 μl.

[0037] Split mode: Splitless injection.

[0038] Solvent delay time: 5 min.

[0039] Column temperature: The initial column temperature was 60 °C; it was increased to 125 °C at a rate of 8 °C / min; then to 210 °C at a rate of 4 °C / min; then to 270 °C at a rate of 5 °C / min; and finally to 305 °C at a rate of 10 °C / min and held for 3 min.

[0040] Mass spectrometry conditions: EI ionization source, ion source temperature 230 °C, quadrupole temperature 150 °C, ionization energy 70 eV, full scan mode (SCAN) was used, and the m / z scan range was 50 - 500.

[0041] During the injection process, one control sample was inserted every 10 injections to confirm the stability of the instrument throughout the injection process.

[0042] 4 Data processing

[0043] The data obtained by GC-MS was compared with the NIST (National Institute of Standards and Technology) database to identify compounds, and then the relative content of the compounds was calculated based on the peak area. The change rate was calculated according to the following formula.

[0044] Change rate = (Relative content of metabolites in the soil of the roots of declining bayberries - Relative content of metabolites in the soil of the roots of healthy bayberries) / Relative content of metabolites in the soil of healthy bayberries × 100%.

[0045] SPSS 17.0 software was used for significance analysis.

[0046] 5 Experimental results

[0047] The TIC diagrams measured by GC-MS for the soil of the roots of healthy bayberries and declining bayberries are shown respectively as Figure 1 and Figure 2 shown. 42 metabolites were identified in the soil samples of the roots of healthy bayberries and declining bayberries. The heat map comparative analysis is shown as Figure 3 shown, and their names, relative contents, and change rates are shown in Table 1 below.

[0048] Table 1 Relative contents of metabolites in the soil of the roots of bayberries and comparison results

[0049]

[0050]

[0051]

[0052] Remarks: * Indicates that it is significantly higher in declining bayberries than in healthy bayberries, p < 0.05; # Indicates that it is significantly lower in declining bayberries than in healthy bayberries, p < 0.05

[0053] It can be seen from the experimental results that: (1) The palatinitol and cytidine in the root soil of declining Myrica rubra are significantly higher than those of healthy Myrica rubra, while the relative contents of the other 40 metabolites in the root soil of declining Myrica rubra are significantly lower than those of healthy Myrica rubra. (2) The change rates of palatinitol and cytidine are 234.67% and 1007.80% respectively, and the change rates of the other 40 metabolites are between -17.37% and -68.74%. The results show that compared with the root soil of healthy Myrica rubra, the metabolite contents in the root soil of declining Myrica rubra have changed significantly. In particular, the changes of palatinitol and cytidine are the most obvious. Their relative content change trends are different from those of other metabolites and the change degree is the largest. Therefore, it shows that the contents of palatinitol and cytidine in the root soil of Myrica rubra are closely related to the decline disease of Myrica rubra. The significant increase in their contents may indicate the occurrence of the decline disease of Myrica rubra.

[0054] Determination of the Contents of Palatinitol and Cytidine in the Root Soil of Declining Myrica rubra with Different Disease Grades in Example 2

[0055] 1 Experimental Materials

[0056] Select the test orchard in the same way as in Example 1.

[0057] Select 6 Myrica rubra trees with decline disease grades of 0 (healthy Myrica rubra), 1, 3, 5, 7, and 9 in the test orchard as test trees.

[0058] The method for collecting root soil is the same as that in Example 1.

[0059] 2 Sample Preparation

[0060] (1) Pretreatment Weigh 1 g of soil sample, add internal standard (20 μl of 0.3 mg / ml L-2-chlorophenylalanine), add 1 ml of methanol-water (v / v = 1:1) and 2 small steel balls. Grind at a frequency of 60 Hz for 2 min. Transfer to a centrifuge tube and centrifuge at 12000 rpm for 10 min. Take 300 μl of the supernatant into a derivatization tube and freeze-dry to obtain powder.

[0061] Take appropriate amounts of palatinitol and cytidine, dissolve and dilute them with methanol-water (v / v = 1:1). Take 1 ml and add internal standard. Take 300 μl into a derivatization tube and freeze-dry to obtain powder. The subsequent treatment is the same as that of the soil sample, which is used as the control sample and the reference.

[0062] (2) Derivatization: Add 80 μl of 15 mg / ml methoxylamine hydrochloride (CNW Technologies GmbH) pyridine solution to the derivatization tube, shake at 37 °C for 90 min to fully carry out the oximation reaction. After drying with nitrogen, add 80 μl of BSTFA (containing 1% TMCS, CNW Technologies GmbH) and 20 μl of n-hexane, incubate at 70 °C for 60 min to fully carry out the silylation reaction.

[0063] 3 GC-MS Chromatographic Conditions

[0064] Under the mass spectrometry conditions, the selected ion mode (SIM) was adopted, and the characteristic fragment ions of palatinose, cytidine, and L-2-chlorophenylalanine were selected for detection. The other conditions were the same as those in Example 1.

[0065] 4 Data Processing

[0066] The contents of palatinose and cytidine in the samples were calculated by the external standard method based on the peak area.

[0067] SPSS 17.0 software was used for significance analysis.

[0068] 5 Experimental Results

[0069] The retention times of the palatinose peak and the cytidine peak in the control chromatogram were consistent with those in the sample chromatogram.

[0070] The contents of palatinose and cytidine in the root zone soil of declining bayberries with different disease grades and the comparison results with the root zone soil of healthy bayberries are shown in Table 2 below.

[0071] Table 2 Contents of palatinose and cytidine in the root zone soil of declining bayberries with different disease grades and comparison results with healthy bayberries

[0072]

[0073] Remarks: * Indicates a significant difference compared with grade 0, p < 0.05; the change rate is the content change rate compared with grade 0.

[0074] It can be seen from the test results that: (1) Compared with the root zone soil of healthy bayberries (grade 0), the contents of palatinose and cytidine in the root zone soil of declining bayberries with different disease grades (1 - 9) were significantly increased (p < 0.05). (2) As the disease grade of declining bayberries increased, the contents of palatinose and cytidine in the root zone soil of bayberries showed an upward trend. This result indicates that there are significant differences in the contents of palatinose and cytidine in the root zone soil of different declining bayberries compared with those of healthy bayberries, and the more severe the disease, the greater the difference. Therefore, the contents of palatinose and cytidine in the root zone soil can be used as diagnostic indicators for bayberry decline disease, and the higher the content, the more severe the disease.

[0075] Example 3: Contents of palatinose and cytidine in rhizosphere soil for diagnosing suspected decline disease of Myrica rubra

[0076] 1 Test method

[0077] In December, three Myrica rubra orchards with decline disease in Haining City, Zhejiang Province (denoted as 1, 2, and 3 respectively) were randomly selected. In the orchards, Myrica rubra trees with fallen mature leaves and weak tree vigor were selected as suspected diseased test trees. Three Myrica rubra trees with dense leaves and healthy tree vigor in the test orchard were selected as healthy test trees. Three suspected diseased test trees were selected from each orchard (marked as 1-D1, 1-D2, 1-D3 in Orchard 1; marked as 2-D1, 2-D2, 2-D3 in Orchard 2; marked as 3-D1, 3-D2, 3-D3 in Orchard 3) and three healthy test trees (marked as 1-H1, 1-H2, 1-H3 in Orchard 1; marked as 2-H1, 2-H2, 2-H3 in Orchard 2; marked as 3-H1, 3-H2, 3-H3 in Orchard 3). The method for collecting rhizosphere soil was the same as that in Example 1. The method for determining the contents of palatinose and cytidine in rhizosphere soil was the same as that in Example 2.

[0078] In June-July of the second year when Myrica rubra was ripe, the fruiting conditions of the above test trees were observed and recorded.

[0079] 2 Test results

[0080] The determination results of the contents of palatinose and cytidine in rhizosphere soil of each Myrica rubra fruit tree and the fruit conditions are shown in Table 3 below.

[0081] Table 3 Determination results of the contents of palatinose and cytidine in rhizosphere soil of Myrica rubra fruit trees and fruit conditions

[0082]

[0083] Remarks: * Indicates significant difference compared with healthy test trees in the same orchard, p<0.05. ns Indicates no significant difference compared with healthy test trees in the same orchard.

[0084] The experimental results show that the contents of palatinose and cytidine in the root soil of suspected diseased bayberries are significantly higher than those of healthy bayberries, and they all show the symptoms of weak disease with large fruit yields and small fruits; for those with no significant difference in the contents of palatinose and cytidine in the root soil from that of healthy bayberries, their fruits are normal and do not show the symptoms of weak disease. The test results confirm that the method of measuring and comparing the contents of palatinose and cytidine in the root soil of suspected diseased and healthy bayberries can be used for the diagnosis of bayberry weak disease, and this method can be used at any time period. Especially before the bayberries bear fruit, early diagnosis and early treatment can be carried out, thus further reducing the losses caused by weak disease bayberries after fruiting.

Claims

1. A method for auxiliary diagnosis of Myrica rubra decline disease by the contents of palatinose alcohol and cytidine in root soil, characterized in that, The method includes: collecting the root zone soils of healthy and suspected diseased Myrica rubra in the same area; measuring the contents of palatinose and cytidine in the root zone soils of healthy and suspected diseased Myrica rubra and conducting comparative analysis; if the contents of palatinose and cytidine in the root zone soil of the suspected diseased Myrica rubra are both significantly increased compared with that of the healthy Myrica rubra, it indicates that the suspected diseased Myrica rubra has a high possibility of getting decline disease; if there is no significant difference in the contents of palatinose and cytidine in the root zone soil of the suspected diseased Myrica rubra, it indicates that the suspected diseased Myrica rubra does not get decline disease; the specific steps for measuring the contents of palatinose and cytidine in the root zone soils of healthy and suspected diseased Myrica rubra include: (1) Take the root zone soil, add methanol-water according to a material-liquid ratio of 1:1, add L-2-chlorophenylalanine as an internal standard, grind at 60 Hz for 2 min, centrifuge, take the supernatant, freeze-dry to obtain a powder for standby; (2) Take the powder, add an excessive amount of methoxyamine hydrochloride pyridine solution, shake at 37 °C for 90 min, dry with nitrogen, add a 4V BSTFA solution containing 1% TMCS and a hexane solution, react at 70 °C for 60 min to obtain a silylated sample solution; (3) Inject the silylated sample solution into a GC-MS for detection, compare the data obtained by GC-MS with the NIST database to identify compounds, then calculate the relative content of the compounds according to the peak area, and calculate the change rate.

2. The method for auxiliary diagnosis of bayberry decline disease based on the contents of palatinose and cytidine in root system soil according to claim 1, characterized in that, The root zone soil is the surface soil of 0-20 cm collected around the drip line of the tree crown.

3. The method for auxiliary diagnosis of Myrica rubra decline disease according to claim 1 by the contents of palatinose and cytidine in root system soil, characterized in that, The area includes Haining City, Zhejiang Province.

4. Use of the contents of palatinose alcohol and cytidine in the root system soil for the diagnosis of bayberry decline disease, characterized in that, The method for diagnosing Myrica rubra decline disease includes: collecting the root zone soils of healthy and suspected diseased Myrica rubra in the same area; measuring the contents of palatinose and cytidine in the root zone soils of healthy and suspected diseased Myrica rubra and conducting comparative analysis; if the contents of palatinose and cytidine in the root zone soil of the suspected diseased Myrica rubra are both significantly increased compared with that of the healthy Myrica rubra, it indicates that the suspected diseased Myrica rubra has a high possibility of getting decline disease; if there is no significant difference in the contents of palatinose and cytidine in the root zone soil of the suspected diseased Myrica rubra, it indicates that the suspected diseased Myrica rubra does not get decline disease; the specific steps for measuring the contents of palatinose and cytidine in the root zone soils of healthy and suspected diseased Myrica rubra include: (1) Take the root zone soil, add methanol-water according to a material-liquid ratio of 1:1, add L-2-chlorophenylalanine as an internal standard, grind at 60 Hz for 2 min, centrifuge, take the supernatant, freeze-dry to obtain a powder for standby; (2) Take the powder, add an excessive amount of methoxyamine hydrochloride pyridine solution, shake at 37 °C for 90 min, dry with nitrogen, add a 4V BSTFA solution containing 1% TMCS and a hexane solution, react at 70 °C for 60 min to obtain a silylated sample solution; (3) Inject the silylated sample solution into a GC-MS for detection, compare the data obtained by GC-MS with the NIST database to identify compounds, then calculate the relative content of the compounds according to the peak area, and calculate the change rate.

5. Use of the contents of palatinose alcohol and cytidine in root system soil in the diagnosis of bayberry decline disease according to claim 4, characterized in that, The root zone soil is the surface soil of 0-20 cm collected around the drip line of the tree crown.

6. Use of the contents of palatinose alcohol and cytidine in root system soil for diagnosing bayberry decline disease according to claim 4, characterized in that, The area includes Haining City, Zhejiang Province.

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