LU501594A1 - Method for identifying walnut disease resistance through walnut leaf stomatal density - Google Patents
Method for identifying walnut disease resistance through walnut leaf stomatal density Download PDFInfo
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- LU501594A1 LU501594A1 LU501594A LU501594A LU501594A1 LU 501594 A1 LU501594 A1 LU 501594A1 LU 501594 A LU501594 A LU 501594A LU 501594 A LU501594 A LU 501594A LU 501594 A1 LU501594 A1 LU 501594A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N21/8422—Investigating thin films, e.g. matrix isolation method
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N2021/8466—Investigation of vegetal material, e.g. leaves, plants, fruits
Abstract
The present disclosure provides a method for identifying walnut disease resistance through stomatal density of walnut leaves. The present disclosure effectively improves the early selection efficiency of walnut hybrid seedlings and accelerates the breeding process of walnut; The present disclosure can eliminate the disease resistant single plant and reduce the breeding cost through the number of stomatal density; in addition, other plant breeding can refer to this method to identify the disease resistance of the plant.
Description
METHOD FOR IDENTIFYING WALNUT DISEASE RESISTANCE THROUGH LU501594
[01] The present disclosure relates to the technical field of plant planting, in particular to a method for identifying walnut disease resistance through walnut leaf stomatal density.
[02] Walnut bacterial black spot and other diseases are severe diseases in walnut producing areas in China, which can cause 30%-50% fruit drop, reduce yield and influent economic benefits. In the process of walnut breeding, cultivating varieties with strong disease resistance is one of the important goals of walnut breeding. However, conventional breeding is a relatively lengthy process, and it takes 16-20 years, sometimes even longer, to breed an ideal variety in the case the hybrid parents are determined. Therefore, early identification of varieties is particularly important. Although the development of molecular-assisted breeding technology has provided the possibility for early selection of hybrids, it has not been widely used due to its high cost and underdeveloped technology.
[03] The natural openings on the surface of plants include stomata, lenticels, water holes, etc. Most bacteria and fungi can invade through the natural openings. In the deconstructed structure of the leaf, there are many stomata in the leaf epidermis scattered among the epidermal cells. The stomata are enclosed by two kidney-shaped guard cells. The stomata space between the two guard cells is called the stomata, and they are the channels for gas exchange between the leaf and the external environment. The number, location and distribution of stomata on the epidermis vary with plant species and are related to ecological conditions. Most plants have an average of 100-300 stomata per square millimeter of the lower epidermis.
[04] There is a certain correlation between plant disease resistance and leaf stomata density. For example, if the stomata density on cucumber leaves is low, the resistance to downy mildew is strong, and vice versa. Studies have shown that downy mildew-resistant grape varieties have significantly fewer leaf stomata than less-resistant varieties. The leaf stomatal density of Fuji apple, pumpkin, alfalfa and other plants was significantly negatively correlated with disease resistance. The higher the stomatal density, the weaker the disease resistance.
[05] Early identification of varieties is an important factor affecting the breeding process and breeding efficiency. Through early identification and selection of the cultivated hybrid seedlings, individual plants with excellent comprehensive characters can be retained, and individual plants with poor characters can be eliminated. This will improve breeding efficiency and reduce the cost of breed development. LU501594
[06] The present disclosure solves the problems in the background technology by providing a method for identifying walnut disease resistance through walnut leaf stomatal density.
[07] In order to solve the above technical problem, the present disclosure employs the following technical solution: a method for identifying walnut disease resistance through walnut leaf stomatal density, comprises the following steps:
[08] Step 1) Collecting walnut leaves of different varieties;
[09] Step 2) Preparing the lower epidermis of the different varieties of walnut leaves into slices;
[10] Step 3) Microscopically observing the number of stomata on the slices;
[11] Step 4) Recording and analyzing the number of stomata to obtain leaf stomatal density of different walnut varieties;
[12] Step 5) Performing correlation analysis on the stomatal density data of different walnut varieties to obtain the correlation between the stomatal density and disease resistance of the different walnut varieties;
[13] Step 6) Determining the ability of the disease resistance of the different walnut varieties according to the correlation between the stomatal density of the different walnut varieties and the disease resistance and the leaf stomatal density of the different walnut varieties.
[14] As a further scheme of the present disclosure: the collection of walnut leaves in the step 1) is obtained by the following method: in the walnut growing season, when the leaves are fully developed, collecting peripheral leaf in the middle and upper parts of the walnut tree in four directions, east, west, north and south. Collecting 5 leaflets in the middle of the pinnate compound leaves in different directions per tree; the collection time is the same each time; washing the collected leaves with water, and blotting the water on the surface of the leaves with paper towels for use.
[15] As a further scheme of the present disclosure: in step 2), preparing the lower epidermis of the different varieties of walnut leaves into slices, employing the collodion method: randomly smearing 5 positions of the lower epidermis with collodion, and after the collodion is dried, removing the collodion sheet with tweezers, placing it on a glass slide, flattening it with a cover glass for later use.
[16] As a further scheme of the present disclosure: the operation steps of step 3) are as follows: observing and photographing the obtained slices under a 400X compound microscope; microscopically observing, randomly selecting 5 horizons, observing the number of stomata N (number) in each horizon with a microscopic imaging system, and measuring the horizon area S
(mm?). LU501594
[17] Asa further scheme of the present disclosure: the operation steps of step 4) are as follows: recording and summarizing the obtained data, calculating the leaf stomatal density per unit area of leaves of different walnut varieties according to formula (1), and obtaining the leaf stomatal density of different walnut varieties (Table 1);
[18] P=N/S(1)
[19] In formula (1): P—stomatal density (unit/mm?); N—stomatal number (number); S—field of view area (mm?).
[20] Table 1 Leaf stomatal density of different walnut varieties Serial Variety Average Horizon area Leaf stomatal numbe number of (mm?) density (unit/mm?) stomata in leaves (unit) BS No.1’ 0.159 125.80 | 2 [Haoning No.4’ 0.159 76.74 | 3 [Vinonine No.5’ 0.159 70.46 | 4 [Haoning No.6’ 0.159 130.82 | 5 [Haoning No.7’ 0.159 62.90 | 6 [Haoning No.10’ 0.159 80.52 Ce No .2’ 0.159 75.50
[21] As a further scheme of the present disclosure: the operation steps of step 5) are as follows: the obtained data is applied to SPSS software for correlation analysis, and its calculation principle is formula (2), and the degree of correlation between stomatal density and disease resistance is determined by the results obtained by the analysis.
2e Ay 3)
[22]
[23] In formula (2): r is the correlation coefficient; x and y are the mean values of the variables x and y, respectively, and x; and y; are the i-th observed values of the variables x and y, respectively. In the present disclosure, x and y respectively represent the stomatal density and diseased fruit rate of different varieties, and x; and yi respectively represent the stomatal density and diseased fruit rate of eight walnut varieties.
[24] In the survey of diseased fruit rate of different varieties, randomly select 5 trees for each variety, and randomly mark each tree with 50 fruits after physiological fruit drop. The number of diseased fruits is investigated in July, and the diseased fruit rate is calculated according to formula (3).
[25] Diseased fruit rate (%) = number of diseased fruit/50x100 (3)
[26] According to the disease resistance and disease rate of different walnut varieties (Table 2), five varieties of 'Liaoning No. 2', 'Liaoning No. 5', 'Hanfeng', 'Liaoning No. 7' and 'Liaoning No. 4' have less than 10% diseased fruits, the disease resistance is strong, and the stomatal density of leaves is less than 80/mm?* The diseased fruit rate of Liaoning No. 10' is within 10-20%, the disease resistance is medium, and the stomatal density is 80.52 /mm?; the diseased fruit rate of "Liaoning No. 1' and 'Liaoning No. 6' are both more than 20% not resistant to disease, stomatal density greater than 125/mm?.
[27] Table 2 Disease resistance and disease rate of different walnut varieties Serial |Variety Average number of Diseased fruitDisease resistance ability SARE cl ee Fm | ww es [mme | ww | wr eee mr | ww | i pen
[28] As a further scheme of the present disclosure: the operation steps of step (6) are as follows: the varieties with a stomatal density P>125/mm? are non-disease-resistant, the variety with P between 80-125/mm° is medium disease-resistant, the varieties with P<80 /mm? are disease- resistant.
[29] Compared with the prior art, the beneficial effects of the present disclosure are as follows: the present disclosure provides a method for identifying disease resistance of walnut through the stomatal density of walnut leaves, the present disclosure effectively improves the early selection efficiency of walnut hybrid seedlings, and accelerates walnut breeding process; the present disclosure can eliminate non-disease-resistant plants through the stomatal density and reduce the breeding cost; in addition, other plant breeding can refer to this method to identify the disease resistance of plants.
[30] Figure 1 is a microscopic photo of the lower epidermis of the leaf of Liaoning No. 7' in the present disclosure, with a magnification of 400X and a horizon area of 0.159 mm”.
[31] Figure 2 is a microscopic photo of the lower epidermis of the leaf of Liaoning No. l'in the present disclosure, with a magnification of 400X and a horizon area of 0.159 mm”.
[32] The technical solutions of the present disclosure will be further explained and illustrated below through specific embodiments.
[33] The leaf stomatal density of walnut varieties in the scientific research base of Liaoning Provincial Economic Forest Research Institute in Songmudao Village, Paotai Town, Jinpu New District, Dalian City was observed.
[34] Example 1
[35] Investigation on the disease rate of different walnut varieties in the field.
[36] (1) Investigated varieties: 'Liaoning No. 1', 'Liaoning No. 4', 'Liaoning No. 5', 'Liaoning No. 6', Liaoning No. 7', "Liaoning No. 10', 'Hanfeng', ' Lipin No. 2"".
[37] (2) Investigation method, 5 trees of each variety were randomly selected, and each tree was randomly marked with 50 fruits after physiological fruit drop.
[38] (3) Date of disease investigation: July 24, 2020.
[39] (4) Calculation method of fruit disease rate: disease rate (%) = number of diseased fruits/50x 100, the disease rate of each walnut variety was counted and analyzed by SPSS. The results showed that (Table 3) there were significant differences in the rate of diseased fruit among 501594 different varieties, indicating that the disease resistance of each variety was different. 'Lipin No. 2' showed the most disease resistance, followed by 'Liaoning 5' and 'Hanfeng', and the least resistant varieties were 'Liaoning 1' and 'Liaoning 6'.
[40] Table 3 Disease rate of different walnut varieties cr © | (Note: In Duncan's test, different lowercase letters in the same column indicate significant differences at the 0.05 level. As long as they contain the same letter, it indicates that there is no significant difference between the two groups.)
[41] Example 2
[42] Determination of stomatal density in leaves of different walnut varieties.
[43] (1) Sampling varieties: the same as those in Example 1.
[44] (2) Sampling date: June 15, 2020. Peripheral leaves of the middle and upper parts of the walnut tree in four directions, east, west, north and south were collected. From each tree, 5 leaflets from the middle part of the pinnate compound leaves in different directions were collected. The collected leaves were washed with water, and the surface of the leaves was dried with paper towels. The lower epidermis of the collected walnut leaves were prepared into slices, and obtained by adopting the collodion method: 5 parts of the lower epidermis was randomly smeared with collodion, and after the collodion dried, the collodion sheet was removed with tweezers, placed on a glass slide, flattened with a coverslip, and set aside.
[45] (3) Microscopic observation method: observe and photograph the obtained sections under a 400X compound microscope; for microscopic observation, 5 horizons were randomly selected,
and the number of stoma N in each horizon was observed with a microscopic imaging system, and, 501594 the horizon area S was measured. The stomatal density P was calculated, the horizon area was
0.159mm?. The stomatal densities of each walnut cultivar were counted and analyzed for significance by SPSS, and the results showed that there were significant differences in leaf stomatal densities among different cultivars (Table 4), 'Liaoning No. 1' (Fig. 1) and 'Liaoning No. 6' had the highest stomata density, with 125.8 /mm?and 130.82 /mm?. 'Liaoning No. 7' (Fig. 2) had the lowest, with 62.9 /mm?.
[46] Table 4 Leaf stomatal density of different walnut varieties Serial Variety Leaf stomatal density (unit/mm?) [Rank number |! [Liaoning No.1’ 125.80 a | 2 [Liaoning No.4’ 76.74 bc | 3 [Leonie No.5’ 70.46 bc | 4 [Leonie No.6’ 130.82 a RE EE No.7’ 62.90 Cc | © [Leonie No.10’ 80.52 b RE (Note: In Duncan's test, different lowercase letters in the same column indicate significant differences at the 0.05 level. As long as they contain the same letter, it indicates that there is no significant difference between the two groups.)
[47] The varieties with a stomatal density P>125/mm? are non- Disease resistant, the variety with P between 80-125/mm? is medium disease-resistant, and the varieties with P<80/mm? are disease-resistant.
[48] Example 3
[49] Correlation analysis between fruit field disease incidence and leaf stomatal density of different walnut cultivars.
[50] The data of Example 1 and Example 2, that is, the leaf stomatal density and the diseased fruit rate, were analyzed by SPSS to determine the correlation between the stomatal density and the diseased fruit rate.
[51] Table 5 Correlation between leaf stomatal density and diseased fruit rate of different walnut varieties LU501594 / Leaf stomatal [Diseased fruit pm Leaf stomatal density Significance bilateral) | | 0.001 N 0 8 08 Secs Gwe) 000 ws (*Significantly correlated at the 0.05 level (bilateral).)
[52] The results showed (Table 5) that there was a significant positive correlation between the field disease incidence of different walnut fruits and the stomatal density of their leaves, and the correlation coefficient was 0.938. It shows that the higher the stomatal density of leaves, the higher the disease rate of walnut fruit.
[53] In summary, through Example 1, Example 2 and Example 3, it can be concluded that there are significant differences in leaf stomatal density and diseased fruit rate among different walnut varieties, and there is a significant positive correlation between stomatal density and diseased fruit rate. There is a regular that the higher the leaf stomatal density, the higher the diseased fruit rate, and the stomatal density per leaf of the disease-resistant varieties is smaller than that of the non- resistant varieties. For example, the stomatal density of leaves of Liaoning No. 7' is significantly lower than that of "Liaoning No. 1', and the diseased fruit rate of Liaoning No. 7' is lower than that of 'Liaoning No. 1'. The experimental results show that the method of observing the stomatal density of walnut can indirectly identify the ability of walnut disease resistance.
Claims (3)
1. A method for identifying walnut disease resistance through walnut leaf stomatal density, wherein, comprises the following steps: step 1) collecting walnut leaves of different varieties; step 2) preparing the lower epidermis of the different varieties of walnut leaves into slices; step 3) microscopically observing the number of stomata on the slices; step 4) recording and analyzing the number of stomata to obtain leaf stomatal density of different walnut varieties; step 5) performing correlation analysis on the stomatal density data of different walnut varieties to obtain the correlation between the stomatal density and disease resistance of the different walnut varieties; step 6) determining the ability of the disease resistance of the different walnut varieties according to the correlation between the stomatal density of the different walnut varieties and the disease resistance and the leaf stomatal density of the different walnut varieties.
2. The method for identifying walnut disease resistance through walnut leaf stomatal density according to claim 1, wherein, in step 2), preparing of the lower epidermis of the different varieties of walnut leaves into slices, employing the collodion method: randomly smearing 5 positions of the lower epidermis with collodion, and after the collodion is dried, removing the collodion sheet with tweezers, placing it on a glass slide, flattening it with a cover glass for later use.
3. The method for identifying walnut disease resistance through walnut leaf stomatal density according to claim 2, wherein the operation steps of step 3) are as follows: observing and photographing the obtained slices under a 400X compound microscope; microscopically observing, randomly selecting 5 horizons, observing the number of stomata N in each horizon with a microscopic imaging system, and measuring the horizon area S.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111664286.XA CN114323865A (en) | 2021-12-31 | 2021-12-31 | Method for identifying disease resistance of walnuts through pore density of walnut leaves |
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| Publication Number | Publication Date |
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| LU501594A1 true LU501594A1 (en) | 2022-09-05 |
| LU501594B1 LU501594B1 (en) | 2022-11-17 |
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| CN (1) | CN114323865A (en) |
| LU (1) | LU501594B1 (en) |
| NL (1) | NL2031150B1 (en) |
| ZA (1) | ZA202202390B (en) |
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|---|---|---|---|---|
| CN106770404B (en) * | 2016-12-01 | 2019-04-09 | 重庆市农业科学院 | A method of identification blade construction and root system root hair whether viral diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106770404B (en) * | 2016-12-01 | 2019-04-09 | 重庆市农业科学院 | A method of identification blade construction and root system root hair whether viral diseases |
Non-Patent Citations (2)
| Title |
|---|
| COSTA LUCAS ET AL: "Determining leaf stomatal properties in citrus trees utilizing machine vision and artificial intelligence", PRECISION AGRICULTURE, SPRINGER US, BOSTON, vol. 22, no. 4, 16 November 2020 (2020-11-16), pages 1107 - 1119, XP037492987, ISSN: 1385-2256, [retrieved on 20201116], DOI: 10.1007/S11119-020-09771-X * |
| EL-SHARKAWY M A: "International research on cassava photosynthesis, productivity, eco-physiology, and responses to environmental stresses in the tropics", PHOTOSYNTHETICA ; INTERNATIONAL JOURNAL FOR PHOTOSYNTHESIS RESEARCH, KLUWER ACADEMIC PUBLISHERS, DO, vol. 44, no. 4, 1 December 2006 (2006-12-01), pages 481 - 512, XP019437106, ISSN: 1573-9058, DOI: 10.1007/S11099-006-0063-0 * |
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| Publication number | Publication date |
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| LU501594B1 (en) | 2022-11-17 |
| ZA202202390B (en) | 2022-04-28 |
| NL2031150B1 (en) | 2023-07-07 |
| CN114323865A (en) | 2022-04-12 |
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