Method for rapidly sorting highly susceptible or highly resistant pine seedlings to Bursaphelenchus xylophilus based on accelerating liquid
By inoculating pine nematodes with a specific ratio of acceleration fluid and artificial skin junction, the process of pine disease sensing is significantly accelerated, and the problem of unstable effect of screening pine resistance varieties in the prior art is solved, and rapid and accurate sorting of high-resistant or high-sensitivity pine germplasm is achieved.
Patent Information
- Application Number
- CN202310089698.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-09
- Publication Date
- 2025-05-27
- Estimated Expiration
- 2043-02-09
AI Technical Summary
The prior art is difficult to quickly and effectively screen out pine varieties with high resistance or high sensitivity of pine nematode disease, resulting in unstable results in the prevention and treatment of pine nematode disease.
The acceleration fluid consisting of NO donors SNP, H2O2, Ca(NO2)2·H2O and MES was used to inoculate the pine nematode suspension by artificial skin junction, and the acceleration fluid was sprayed after inoculation to observe the onset time and sensitivity of the plants, and the high-sensitivity or high-resistant pine seedlings were sorted.
The pine tree germ disease process has been significantly accelerated, the stability and repetition of the inoculation effect have been improved, and the high resistance or high sensitivity of pine tree germplasm can be quickly and accurately screened out.
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Figure CN116369106B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a method for rapidly sorting highly susceptible or highly resistant pine seedlings to Bursaphelenchus xylophilus based on an accelerating solution.
Background Art
[0002] Pine wilt disease is a devastating forest disease caused by the parasitic Bursaphelenchus xylophilus in pine trees, known as the "cancer of pine trees". Pine trees are the main tree species for ecological construction and afforestation in southern China and play a crucial role in the development of the forestry economy. In recent years, the damage of pine wilt disease has been extremely serious, with a high mortality rate of infected plants and extremely rapid spread. The affected area in China has exceeded 27 million mu. Since the invasion of pine wilt disease into China more than 30 years ago, billions of pine trees have died, resulting in direct and indirect economic losses of over a thousand billion yuan. A series of methods have been used in China to control pine wilt disease, including felling diseased trees, debarking, burning, fumigation, spraying nematicides, controlling vector insects, improving forest land hygiene, and enhancing the vitality of forest trees, but none of them have been able to effectively prevent and control the occurrence and spread of pine wilt disease fundamentally. If this continues, it will pose a serious threat to China's timber security, ecological security, and economic construction. Therefore, it is urgent to carry out targeted scientific and technological research and breakthroughs in the field of pine wilt disease prevention and control.
[0003] The breeding of resistant tree varieties has become an effective long-term strategy for controlling forest pests and diseases and has received much attention and achieved good results in many countries around the world. Previous studies have shown that there are obvious resistance differences among individual plants within a tree species, and there are even individual plants with extremely high resistance even in susceptible tree species. The growth cycle of pine trees is long, and traditional conventional breeding cannot effectively solve the shortage of excellent germplasms of fast-growing and highly resistant pine trees. Breeding and utilization of excellent resistant germplasms through tissue culture technology is the most effective way to solve the current pine wilt disease in pine trees and achieve sustainable and efficient development of the industry. The research results on the resistance of pine trees to pine wilt disease show that resistant pine trees generally have better growth conditions. Pine trees have a taproot system. It has been found that the root quality of seedlings cultivated through tissue culture breeding is significantly improved, with strong environmental adaptability and outstanding performance in field growth. Using tissue culture seedlings cultivated from adult pine trees with strong growth, high yield, and disease resistance selected from pine wilt disease epidemic areas as test materials, the method of artificially inoculating Bursaphelenchus xylophilus is an effective method for screening excellent resistant germplasms of pine trees. Currently, from the publicly reported literature, the inoculation of Bursaphelenchus xylophilus is easily affected by various biological and abiotic factors, with large fluctuations in the infection rate and mortality rate, low repeatability, and unstable inoculation effects.
[0004] Therefore, it is necessary to develop a method for quickly determining whether a pine tree variety is disease-resistant, so as to sort the pine trees in the wild diseased forest and quickly obtain highly disease-resistant and excellent individual plants with good growth traits and better potential for resistance to pine wilt disease as the breeding objects.
Summary of the Invention
[0005] In view of the above, it is necessary to develop a method for quickly determining whether a pine tree variety is disease-resistant, so as to sort the pine trees in the wild diseased forest and quickly obtain highly disease-resistant and excellent individual plants with good growth traits and better potential for resistance to pine wilt disease as the breeding objects.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] An accelerating solution for accelerating the course of pine trees infected with pine wilt disease, the accelerating solution consists of 50 - 150 μM / L of NO donor SNP, 5% - 15% H 2 O 2 , 1 - 2 mM / L Ca(NO 2 ) 2 ·H 2 O and 3 mM / L MES.
[0008] Further, the pH of the accelerating solution is 6.5.
[0009] The present invention also includes a method for quickly sorting highly susceptible or highly disease-resistant pine tree seedlings with the accelerating solution. The method is as follows: Select adult individual pine trees with a tree age of ≥ 20 years as the breeding objects, obtain pine tree seedlings through stem bud tissue culture, then inoculate the pine tree seedlings with a pine nematode suspension by artificial bark grafting method, spray the accelerating solution on the inoculated plants, and conduct routine management and observation on the plants; when the disease onset time of the plants is ≤ 10 days, and both the disease infection rate and the mortality rate are ≥ 95%, then the plants are sorted as highly susceptible pine wilt disease seedlings; when the disease onset time of the plants is ≤ 10 days and both the disease infection rate and the mortality rate are ≤ 5%, then the plants are sorted as highly disease-resistant pine wilt disease seedlings.
[0010] Further, the spraying times of the accelerating solution are respectively the 3rd, 7th, 15th, 30th, and 50th days after nematode inoculation, and the spraying amount is 100 mL per plant each time.
[0011] Further, the pine nematode suspension is a 15 nematodes / μL pine nematode suspension prepared with 0.5% H 2 O 2 .
[0012] Further, the calculation model of the disease infection rate is: disease infection rate = number of diseased plants / total number of plants × 100%; the calculation model of the mortality rate is: mortality rate = number of dead plants / total number of plants × 100%.
[0013] In this application, the pine tree is Pinus massoniana Lamb.
[0014] The present invention has the following beneficial effects:
[0015] Based on the signal cascade reaction mechanism during the pathogenic process of Bursaphelenchus xylophilus, the present invention explores the effects of nitric oxide (NO), reactive oxygen species (ROS), and calcium ion signals on the inoculation effect of Bursaphelenchus xylophilus. The applicant found that by systematically optimizing the parameters of the above influencing factors, the disease-susceptible process of pine trees is significantly accelerated, with high repeatability and stable inoculation effect, providing strong scientific and technological support for sorting pine germplasms with different resistances. The present invention uses signal substances to prepare an accelerating solution for accelerating the course of pine wilt disease. Through the improvement of the accelerating solution, the onset time of pine wilt disease can be advanced from about 30 days in the conventional method to about 10 days, with very prominent effects. The research and development of this accelerating solution can more accurately screen out plants with more stable disease-susceptible effects. By combining the onset time, disease-susceptible rate, and mortality rate, pine tree plants highly susceptible or highly resistant to Bursaphelenchus xylophilus can be quickly distinguished to obtain corresponding germplasm resources.
Description of the Drawings
[0016] Figure 1 It is a growth trend diagram of seedlings of the same variety on the 10th day after being treated with the accelerating solution and without the accelerating solution; in the figure, A is the seedling treated with the accelerating solution, and B is the seedling not treated with the accelerating solution;
[0017] Figure 2 It is a growth trend diagram of seedlings of the same variety on the 35th day after being treated with the accelerating solution and without the accelerating solution; in the figure, A is the seedling treated with the accelerating solution, and B is the seedling not treated with the accelerating solution;
[0018] Figure 3 It is a growth trend diagram of highly susceptible and highly resistant seedlings on the 55th day after being treated with the accelerating solution; in the figure, A is the highly susceptible seedling, and B is the highly resistant seedling.
Detailed Embodiments
[0019] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.
[0020] Any feature disclosed in this specification (including any additional claims, abstract) is, unless specifically stated, only an example of a series of equivalent or similar features.
[0021] Example 1:
[0022] Based on the signal cascade reaction mechanism during the pathogenic process of Bursaphelenchus xylophilus, this invention explores the effects of nitric oxide (NO), reactive oxygen species (ROS), and calcium ion signals on the inoculation effect of Bursaphelenchus xylophilus, and studies the effects of different signal substance ratios on the disease process of pine trees infected with Bursaphelenchus xylophilus. The specific research methods include the following steps:
[0023] (1) Seedling preparation: Select diseased trees with an age of ≥20 years, obvious disease symptoms, and weak growth vigor screened from the Bursaphelenchus xylophilus disease epidemic area as the breeding objects, and use the stem bud tissue culture method to cultivate seedlings. After 1.5 years of seedling cultivation, select seedlings with a height of ≥40 cm, a diameter of ≥5 mm, and a high degree of lignification at 15 - 20 cm above the stem base as the test materials;
[0024] (2) Nematode treatment: Adopt the H 2 O 2 gradient method. Disinfect the cultured nematodes of the highly pathogenic mixed strain series with 15% → 8% → 4% → 2% H 2 O 2 . After washing with sterile water, make a nematode suspension of about 15 nematodes / μL with 0.5% H 2 O 2 for standby;
[0025] (3) Nematode inoculation: Under sunny weather and an environmental temperature of 30°C, use the artificial bark grafting method. Make an incision with a sterilized small knife at 10 - 12 cm above the stem base. The incision width is 2 - 3 mm, the length is 1.5 - 4 cm, and the depth is 1 mm. After cutting to the xylem, gently scrape with a sterile saw. According to the dosage of 200 μL of nematode suspension per single plant inoculation, put the nematode suspension on medical absorbent cotton with a pipette, and then place it close to the incision and seal it with a sealing film;
[0026] (4) Seedling treatment: Treat the seedlings by foliar spraying with the accelerating solution. The spraying times are the 3rd, 7th, 15th, 30th, and 50th days after nematode inoculation, and the spraying amount is 100 mL per plant each time. The accelerating solution is composed of the NO donor SNP (sodium nitroprusside, molecular formula Na 2 [Fe(CN)5NO]·2H 2 O), 5% - 15% H 2 O 2 , 1 - 2 mM / L Ca(NO 2 ) 2 ·H 2 O, and 3 mM / L MES (morpholineethanesulfonic acid, molecular formula C 6 H 13 NO 4 S), with a pH of 6.5;
[0027] (5) Seedling screening: After inoculating with nematodes, dynamically track and observe the disease symptoms of seedlings such as wilting, water loss, and withering, and select plants with rapid onset (onset time ≤ 10 days) as highly susceptible Pinus massoniana seedlings to Bursaphelenchus xylophilus.
[0028] In step (4), the accelerating liquid that accelerates the course of Bursaphelenchus xylophilus disease in Pinus massoniana is specifically screened and determined for its components using an orthogonal experiment. In step (5), after inoculating with nematodes, in addition to observing the onset time, the disease infection rate and mortality rate of the seedlings are statistically analyzed on the 35th and 55th days.
[0029] Among them, the calculation formulas for the disease infection rate and mortality rate are as follows:
[0030] Disease infection rate = number of diseased plants / total number of plants × 100%;
[0031] Mortality rate = number of dead plants / total number of plants × 100%.
[0032] Specifically, as shown in Table 1:
[0033] Table 1 Observation results of highly susceptible Pinus massoniana nematode inoculation in the orthogonal experiment of the accelerating liquid
[0034]
[0035]
[0036] Note: The values in the table are "mean ± standard deviation", and lowercase letters indicate significant differences between different treatments (p < 0.05).
[0037] As can be seen from Table 1, under different signal substance treatments, there are significant differences in the onset time, disease infection rate, and mortality rate of the seedlings after inoculating with nematodes. Among them, in the treatment without adding signal substances (T1), the onset time is 32.5 days, the disease infection rate and mortality rate are 88.2% and 86.9% respectively, and the standard deviation > 10%. While in the T8, T12, T14, and T15 treatments, the onset time is advanced to 9.1 - 10.0 days, and both the incidence rate and mortality rate exceed 95%, and the standard deviation < 5%. This shows that with 50 - 150 μM / L SNP + 5% - 15% H 2 O 2 + 1 - 2 mM / L Ca(NO 2 ) 2 ·H 2When treated with O + 3 mM / L MES, the onset time of the seedlings' disease significantly accelerated, with obvious disease susceptibility and death characteristics, and high repeatability, greatly improving the efficiency of screening disease-resistant seedlings. In addition, it is worth mentioning that under the T10 treatment, the shortest onset time of the seedlings was only 7.8 days, the incidence and mortality rates were both greater than 95.6%, and the standard deviation was less than 5%. However, its mortality rate was close to 100%, significantly higher than the incidence rate. Signal substances have a dual effect of promoting growth at low concentrations and inhibiting growth at high concentrations on plants. This indicates that the treatment with this signal substance has a synergistic effect of causing certain stress damage to the plants and fails to objectively reflect the resistance of the plants to Bursaphelenchus xylophilus, suggesting that the use concentration of the signal substance must be strictly controlled within an appropriate range.
[0038] Example 2:
[0039] Use the most optimized treatment T14 with the accelerating solution and the treatment T1 without the accelerating solution in the orthogonal table of Example 1 to treat Masson pine seedlings of the same variety. The treatment method is as follows:
[0040] Experimental group: Obtain pine seedlings through stem bud tissue culture of adult trees of the same variety, then inoculate the pine seedlings with the suspension of Bursaphelenchus xylophilus by artificial bark grafting method, spray the accelerating solution treated with T14 on the inoculated plants, and conduct routine management and observation on the plants.
[0041] Control group: Obtain pine seedlings through stem bud tissue culture of adult trees of the same variety, then inoculate the pine seedlings with the suspension of Bursaphelenchus xylophilus by artificial bark grafting method, spray the clear water treated with T1 (without any accelerating solution components) on the inoculated plants, and conduct routine management and observation on the plants.
[0042] The results obtained after 10 days of treatment of the experimental group and the control group are shown in Figure 1 :
[0043] Figure 1 In the figure, A is the Masson pine seedlings of the experimental group, and B is the Masson pine seedlings of the control group. It can be seen from the figure that the needles of the Masson pine in group A are significantly withered and yellowed, while the needles of the Masson pine in group B still remain green.
[0044] The results obtained after 35 days of treatment of the experimental group and the control group are shown in Figure 1 :
[0045] Figure 1 In the figure, A is the Masson pine seedlings of the experimental group, and B is the Masson pine seedlings of the control group. It can be seen from the figure that the needles of the Masson pine in group A are completely withered and yellowed, while the needles of the Masson pine in group B still remain partially green.
[0046] It can be seen from the figure that the accelerating solution has accelerated the disease progression of Bursaphelenchus xylophilus in Masson pine. Figure 1 - Figure 2The disease progression of Group A was significantly faster than that of Group B. This indicates that after applying the accelerating solution, it can cause Masson pine to develop the disease rapidly, enabling a faster screening of the diseased Masson pine plants.
[0047] Example 3:
[0048] Sorting of highly resistant pine wilt disease seedlings:
[0049] Use the four preferred treatments T8, T12, T14, and T15 with the accelerating solution in the orthogonal array of Example 1 to screen for highly resistant pine wilt disease seedlings. The specific method is as follows:
[0050] In a 20 - 25 - year - old Masson pine forest stand in a pine wilt disease epidemic area without human intervention, select three preferred dominant trees with straight trunks, strong growth, and no pests and diseases as the breeding objects and number them as GL - 15, GL - 36, and GL - 112. Obtain tissue - cultured seedlings through stem - bud tissue culture of the breeding objects, and then inoculate the pine nematode suspension onto the seedlings using the artificial bark - grafting method. After inoculation, spray the accelerating solution on the plants (select the four most preferred treatments T8, T12, T14, and T15 in the orthogonal array of Example 1 for the composition of the accelerating solution). Conduct routine management and observation of the plants; when the disease onset time of the plant ≤ 10 days and both the disease incidence rate and the mortality rate ≤ 5%, then select this plant as a highly resistant pine wilt disease seedling; when the disease onset time of the plant ≤ 10 days and both the disease incidence rate and the mortality rate > 5%, then this plant cannot be selected as a highly resistant pine wilt disease seedling. The specific results are shown in Table 2:
[0051] Table 2 Sorting of tissue - cultured seedlings of three groups of superior Masson pine plants for resistance to pine wilt disease
[0052]
[0053] As can be seen from Table 2, the average disease onset time of the three groups of superior plants was < 10 days, reflecting that Masson pine was relatively sensitive to the tested signal substance treatments, indicating that the experimental treatment results were effective. However, considering the disease resistance ability of the plants themselves, the average disease incidence rate and mortality rate of GL - 15 were both > 5%, and the standard deviation of its disease incidence rate > 5%, indicating that the disease resistance ability of the seedlings bred from this plant was unstable. Therefore, it cannot be selected as a highly resistant pine wilt disease seedling; relatively, the average disease incidence rate and mortality rate of GL - 36 and GL - 112 were both ≤ 5%, and their standard deviation values were much lower than 5%, indicating that the seedlings bred from the two genotype superior trees had strong and stable disease resistance ability. Therefore, the clonal seedlings of GL - 36 and GL - 112 were selected as highly resistant pine wilt disease seedlings.
[0054] Several Pinus massoniana varieties in Table 2 were also correspondingly confirmed in the later planting experiments. After tracking and observation, the disease-infected mortality rates of Pinus massoniana GL-15, GL-36, and GL-112 were 10.8%, 4.3%, and 4.8% respectively.
[0055] The results of Comprehensive Examples 1-2 show that the best cultivation method for quickly sorting Pinus massoniana seedlings resistant to Bursaphelenchus xylophilus is as follows:
[0056] (1) Seedling preparation: Select adult single plants with a tree age of ≥ 20 years and obvious disease-infected or disease-resistant characteristics screened from the Bursaphelenchus xylophilus epidemic area as the breeding objects, and use the stem bud tissue culture method to cultivate seedlings. After 1.5 years of seedling cultivation, select seedlings with a height of ≥ 40 cm, a stem diameter of ≥ 5 mm, and a high degree of lignification at 15-20 cm above the stem base as test materials;
[0057] (2) Nematode treatment: Adopt the H 2 O 2 gradient method to serially disinfect the cultivated nematodes of the highly pathogenic mixed strain using 15% → 8% → 4% → 2% H 2 O 2 . After washing clean with sterile water, make a nematode suspension of about 15 nematodes / μL with 0.5% H 2 O 2 for standby;
[0058] (3) Nematode inoculation: Under the condition of fine weather and an environmental temperature of 30 °C, use the artificial bark grafting method. Make an incision with a sterilized small knife at 10-12 cm above the stem base, with a width of 2-3 mm, a length of 1.5-4 cm, and a depth of 1 mm. After cutting to the xylem, gently scrape with a sterile saw. According to the dosage of 200 μL of nematode suspension per single plant inoculation, put the nematode suspension on medical absorbent cotton with a pipette, and then closely attach it to the incision and seal it with sealing film;
[0059] (4) Seedling treatment: Use the accelerating solution to treat the seedlings by foliar spraying. The spraying times are the 3rd, 7th, 15th, 30th, and 50th days after nematode inoculation, and the spraying amount is 100 mL per plant each time. The accelerating solution consists of 50-150 μM / L NO donor SNP (sodium nitroprusside, molecular formula Na 2 [Fe(CN)5NO]·2H 2 O), 5%-15% H 2 O 2 , 1-2 mM / L Ca(NO 2 ) 2 ·H 2 O and 3 mM / L MES (morpholineethanesulfonic acid, molecular formula C 6 H 13 NO 4 S), with a pH of 6.5;
[0060] (6) Seedling screening: After inoculating with nematodes, dynamically track and observe the disease conditions of seedlings such as wilting, water loss, and withering. At the 35th and 55th days after inoculating with nematodes, count the disease incidence rate and mortality rate of the seedlings. When the disease onset time of the plant ≤ 10 days, and both the disease incidence rate and mortality rate ≥ 95%, then select this plant as a highly susceptible pine wood nematode disease seedling; when the disease onset time of the plant ≤ 10 days and both the disease incidence rate and mortality rate ≤ 5%, then select this plant as a highly resistant pine wood nematode disease seedling.
[0061] Verification experiment:
[0062] Adopt the most optimized treatment T14 in the orthogonal array of Example 1 to screen highly susceptible and highly resistant seedlings for tissue culture seedlings of different varieties, and the results are as follows:
[0063] Experimental method: Select adult single plants of pine trees as the breeding objects, obtain pine seedlings through stem bud tissue culture, then inoculate the pine seedlings with pine nematode suspension by artificial bark grafting method, spray the accelerating liquid on the inoculated plants, and conduct routine management and observation on the plants; when the disease onset time of the plant ≤ 10 days, and both the disease incidence rate and mortality rate ≥ 95%, then select this plant as a highly susceptible pine wood nematode disease seedling; when the disease onset time of the plant ≤ 10 days and both the disease incidence rate and mortality rate ≤ 5%, then select this plant as a highly resistant pine wood nematode disease seedling.
[0064] The results are as Figure 3 shown. It can be seen from the figure that A is the seedling of a highly susceptible variety and B is the seedling of a highly resistant variety; at the 55th day, the seedlings of the highly susceptible variety have completely withered, while the seedlings of the highly resistant variety still remain green. It shows that the method of this application is feasible, and the disease-resistant or disease-susceptible characteristics of the selected seedlings are very obvious. Combining with the disease course display effect of Example 2, it can be known that after using the accelerating liquid, the disease manifestation process of Masson pine seedlings after being infected with the disease is accelerated, and within the appropriate dose range, the accelerating liquid will not affect the pine seedlings, will not cause misjudgment, and can greatly shorten the sorting time of seedlings and improve the accuracy of seedling sorting.
[0065] In summary, the present invention prepares an accelerating liquid for accelerating the course of Masson pine infected with pine wood nematode by optimizing the effects of nitric oxide, reactive oxygen species, and calcium ion signals on the inoculation effect of pine wood nematode, which significantly accelerates the disease infection process of pine trees, provides a powerful scientific and technological support for effectively distinguishing highly susceptible or highly resistant plants of Masson pine pine wood nematode disease in the later stage, and for obtaining excellent germplasms of pine trees with different resistances in the future.
[0066] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.
Claims
1. A method for rapidly sorting pine tree seedlings with high resistance or high susceptibility to Bursaphelenchus xylophilus using an acceleration liquid, characterized in that, the method is as follows: Select adult single pine trees as breeding objects, obtain pine tree seedlings through tissue culture, then inoculate the pine tree seedlings with a suspension of pine wood nematodes by artificial bark grafting method, spray the acceleration liquid on the inoculated plants, and conduct routine management and observation on the plants; when the disease onset time of the plants ≤ 10 days, and both the disease infection rate and the mortality rate ≥ 95%, then select the plants as pine tree seedlings with high susceptibility to Bursaphelenchus xylophilus; when the disease onset time of the plants ≤ 10 days and both the disease infection rate and the mortality rate ≤ 5%, then select the plants as pine tree seedlings with high resistance to Bursaphelenchus xylophilus; The accelerating solution consists of 50 - 150 μM / L NO donor SNP, 5% - 15% H 2 O 2 , 1 - 2 mM / L Ca(NO 2 ) 2 ·H 2 O and 3 mM / L MES; the pH of the acceleration liquid is 6.5; the pine tree is Pinus massoniana.
2. The method according to claim 1, characterized in that, the acceleration liquid is sprayed on the 3rd, 7th, 15th, 30th, and 50th days after nematode inoculation, and the spraying amount is 100 mL per plant each time.
3. The method according to claim 1, characterized in that, The pine wood nematode suspension is a 15 nematodes / μL pine wood nematode suspension prepared with 0.5% (volume percentage) H 2 O 2 .
4. The method according to claim 1, characterized in that, the calculation model of the disease infection rate is: disease infection rate = number of diseased plants / total number of plants × 100%; the calculation model of the mortality rate is: mortality rate = number of dead plants / total number of plants × 100%.