Tomato TY virus occurrence early warning method based on temperature and bemisia tabaci population density

Abstract

The invention belongs to the technical field of tomato planting, and particularly relates to a tomato TY virus occurrence early warning method based on temperature and bemisia tabaci population density, which comprises the following steps: establishing a correlativity model of virus gene relative expression quantity Y, temperature x1 and bemisia tabaci population density x2, and measuring the temperature x1 and the bemisia tabaci population density x2 in a tomato planting site; and when the virus gene relative expression quantity Y obtained through the correlation model exceeds an early warning value, triggering a prevention alarm. According to the method, the correlativity model of the temperature and the bemisia tabaci population density and the relative expression quantity of the virus gene of the tomato TY virus is established, the temperature and the bemisia tabaci population density are convenient to measure in a tomato planting site, and the relative expression quantity of the virus gene of the current tomato TY virus can be predicted by means of the correlativity model; a basis is provided for the infection condition of the tomato TY virus, so that growers can prevent, intervene and treat the tomato TY virus in advance, and the tomato planting loss is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of tomato planting, and in particular relates to a tomato TY virus occurrence early warning method based on temperature and whitefly population density. Background Art

[0002] Tomato TY virus (TYLCV) is a devastating plant disease, mainly spread by whiteflies. Tomato plants infected with TY virus will show symptoms such as growth retardation, dwarfing, smaller top leaves, wrinkled clusters, slightly yellowing, and curled edges. After adult plants are infected, only the upper leaves and new shoots show symptoms, while the middle and lower leaves and fruits are generally not affected. The virus has the characteristics of sudden outbreak, rapid spread, and difficulty in treatment, causing serious losses to tomato production. Summary of the invention

[0003] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a tomato TY virus occurrence early warning method based on temperature and whitefly population density, which can predict the outbreak and spread risk of tomato TY virus through parameter changes of temperature and whitefly population density, and provide a reference basis for tomato planting management.

[0004] The specific technical solution adopted by the present invention is:

[0005] A tomato TY virus early warning method based on temperature and whitefly population density was developed. A correlation model among the relative expression of the virus gene Y, temperature x1, and whitefly population density x2 was established. The temperature x1 and whitefly population density x2 in the tomato planting site were measured. When the relative expression of the virus gene Y obtained through the correlation model exceeded the early warning value, a prevention and control alarm was triggered.

[0006] The correlation model is:

[0007] Y=0.01x1 3 -0.94x1 2 +29.1x1+0.85[A2+(A1-A2) / (1+x2 / x0)]-291.08

[0008] in,

[0009] Y: relative expression of viral genes;

[0010] x1: temperature;

[0011] x2: population density;

[0012] A1: 2.32684±3.15992;

[0013] A2: 36.98764±3.15992;

[0014] x0:45.01±6.38415.

[0015] The method for establishing the correlation model comprises the following steps:

[0016] S1. Build cages and cover with insect-proof nets, and plant several tomato seedlings in each cage;

[0017] S2. After planting, select inoculated and non-inoculated strains in the cage, and inoculate the inoculated strains with tomato TY virus;

[0018] S3. After inoculation, whiteflies are placed in the cage according to the set population density;

[0019] Sampling was started on the third day after the release of whiteflies to detect the viral load in the plants not inoculated with the virus. The relative expression level Y of the viral gene in the leaves was analyzed by qPCR, and a correlation model was established based on the greenhouse temperature x1 and the whitefly population density x2 of the corresponding treatment.

[0020] When determining the effect of whitefly population density x2 on tomato TY virus infection, in step S1, 8 1.2×1.5×1.5 meter cages were built in the greenhouse, covered with 60-mesh insect-proof nets, and 12 tomato seedlings were planted in each net cover, arranged in a 4×3 pattern to form a seedling array;

[0021] In the step S2, 10 days after the seedlings are planted, two tomato seedlings in the middle of each seedling array in each cage are used as inoculated plants and inoculated with tomato TY virus;

[0022] In the step S3, whiteflies were placed one week after inoculation of tomato TY virus. The experiment had 7 treatments and 1 control. 0, 50, 100, 200, 400, 600, 800, and 1000 whiteflies were placed respectively, of which 0 was the control group.

[0023] In step S4, 5 plants are randomly selected from the 10 non-inoculated plants at the inner edge of each cage for sampling, as 5 repetitions, and the top leaves are picked for sampling, and the sampling is carried out once every 3 days, for a total of 4 times.

[0024] When measuring the effect of temperature x1 on tomato TY virus infection, in step S1, 6 temperature gradients of 24°C, 26°C, 28°C, 30°C, 32°C, and 34°C are set in the incubator, and a 30×30×30 cm cage is placed in each incubator, and 4 tomato seedlings are placed in each cage;

[0025] In step S2, two diagonal tomato seedlings are used as inoculated plants and inoculated with tomato TY virus;

[0026] In the step S3, one week after the tomato TY virus is inoculated, whiteflies are placed in each cage, and 50 whiteflies are inoculated;

[0027] In the step S4, non-inoculated strains are sampled once every 3 days, for a total of 5 times.

[0028] It also includes a method for culturing tomato TY virus: use an inoculation loop to dip the bacterial liquid from the infectious clone strain preserved at -70°C, streak on YEB solid culture medium, culture at 28°C to obtain colonies, complete the cultivation of tomato TY virus, and use a sterile toothpick to pick up the colonies and smear them on the back of tomato leaves and tender stems for inoculation.

[0029] YEB solid culture medium contains 50ug / mL kanamycin and 50ug / mL rifampicin.

[0030] Before the experiment, tomato seedlings and whiteflies were tested for TYLCV. The detection method was as follows: genomic DNA of tomato seedling leaves and whiteflies was extracted, PCR amplification was performed using TYLCV-specific gene primers TYLCV-61 and TYLCV-473, and the obtained PCR products were detected by 1.2% agarose gel electrophoresis. If no virus-specific gene fragments were amplified, it indicated that the laboratory-cultivated tomatoes and the reared whitefly populations themselves did not carry tomato yellow leaf curl virus, and met the experimental requirements.

[0031] The reaction procedure of the PCR amplification was: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, and extension at 68°C for 1 s, for a total of 35 cycles.

[0032] The sequence of the TYLCV-specific gene primer TYLCV-61 is "5'-ATACTTGGACACCTAATGGC-3'", and the sequence of TYLCV-473 is "5'-AGTCACGGGCCCTTACAA-3'".

[0033] The beneficial effects of the present invention are:

[0034] The present invention uses temperature and whitefly population density to establish a correlation model with the relative expression amount of viral genes of tomato TY virus. In a tomato planting site, temperature and whitefly population density are easy to measure, and the correlation model can be used to predict the current relative expression amount of viral genes of tomato TY virus, providing a basis for the infection status of tomato TY virus.

[0035] The present invention also designs rigorous experimental steps for determining the effects of whitefly population density and temperature on tomato TY virus infection to ensure the accuracy and reliability of the data. When the relative expression amount Y of the virus gene obtained by the correlation model exceeds the warning value, a prevention and control alarm is triggered to remind growers to take precautions, so that growers can take preventive measures, intervene, and treat in advance to reduce tomato planting losses. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The impact of different population sizes of whiteflies on virus transmission;

[0037] Figure 2 The correlation curve model between viral load and whitefly population size;

[0038] Figure 3 Effects of different temperatures on the ability of whiteflies to transmit viruses;

[0039] Figure 4 Correlation curve model between viral load and temperature;

[0040] Figure 5 Electrophoresis of PCR amplification of Tomato Yellow Leaf Curl Virus (TYLCV); DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments:

[0042] Specific implementation examples Figure 1 As shown, the tomato TY virus occurrence early warning method based on temperature and whitefly population density is established. A correlation model among the relative expression of the virus gene Y, temperature x1 and whitefly population density x2 is established, and the temperature x1 and whitefly population density x2 in the tomato planting site are measured. When the relative expression of the virus gene Y obtained by the correlation model exceeds the early warning value, a prevention and control alarm is triggered.

[0043] The correlation model is:

[0044] Y=0.01x1 3 -0.94x1 2 +29.1x1+0.85[A2+(A1-A2) / (1+x2 / x0)]-291.08

[0045] in,

[0046] Y: relative expression of viral genes;

[0047] x1: temperature;

[0048] x2: population density;

[0049] A1: 2.32684±3.15992;

[0050] A2: 36.98764±3.15992;

[0051] x0:45.01±6.38415.

[0052] The method for establishing the correlation model comprises the following steps:

[0053] S1. Build cages and cover with insect-proof nets, and plant several tomato seedlings in each cage;

[0054] S2. After planting, select inoculated and non-inoculated strains in the cage, and inoculate the inoculated strains with tomato TY virus;

[0055] S3. After inoculation, whiteflies are placed in the cage according to the set population density;

[0056] Sampling was started on the third day after the release of whiteflies to detect the viral load in the plants not inoculated with the virus. The relative expression level Y of the viral gene in the leaves was analyzed by qPCR, and a correlation model was established based on the greenhouse temperature x1 and the whitefly population density x2 of the corresponding treatment.

[0057] Experiment 1: When determining the effect of whitefly population density x2 on tomato TY virus infection

[0058] In the step S1, eight 1.2×1.5×1.5 meter cages are built in the greenhouse, covered with 60-mesh insect-proof nets, and 12 tomato seedlings are planted in each net cover, arranged in a 4×3 pattern to form a seedling array;

[0059] In the step S2, 10 days after the seedlings are planted, two tomato seedlings in the middle of each seedling array in each cage are used as inoculated plants and inoculated with tomato TY virus;

[0060] In the step S3, whiteflies were placed one week after inoculation of tomato TY virus. The experiment had 7 treatments and 1 control, and 0, 50, 100, 200, 400, 600, 800, and 1000 whiteflies were placed respectively, of which 0 was the control group. In order to verify the correlation between whiteflies and the occurrence and spread of tomato TY virus, a T0 control group was set, and the T0 control group included multiple blank control groups. The tomato plants in each blank control group had the same layout as other experimental groups, and 0, 50, 100, 200, 400, 600, 800, and 1000 whiteflies were placed respectively, but the tomato TY virus was not inoculated;

[0061] In step S4, 5 plants are randomly selected from the 10 non-inoculated plants at the inner edge of each cage for sampling, as 5 repetitions, and the top leaves are picked for sampling, and the sampling is carried out once every 3 days, for a total of 4 times.

[0062] qPCR analysis was performed on tomato leaf samples collected at different times after inoculation with whiteflies. Figure 1 The viral load in tomato plants inoculated with 50, 100, 200, and 400 whiteflies began to increase from the sixth day after inoculation, but the rate of increase was slow, and no virus was detected in the T0 control group; the viral load in tomato plants inoculated with 600, 800, and 1000 whiteflies began to increase significantly from the sixth day after inoculation, reaching a peak on the ninth day, and the viral content decreased significantly on the 12th day. 3 )-treated tomato plants showed the highest viral load 9 days after whitefly inoculation.

[0063] No virus was detected in the T0 control group during the entire experiment. Based on the experimental results, it can be inferred that there is a direct correlation between the spread of whiteflies and TY virus. In the experimental group, the virus load in tomato leaves showed an overall trend of first increasing and then decreasing; when the whitefly population density was low, the virus spread slowly. Only when the whitefly population density in the cage exceeded 400 (148 heads / m 3 ) will have a greater impact on the spread of the virus.

[0064] The relationship between the whitefly population density and the relative expression of the TYLCV gene on the 9th day was fitted and analyzed. The results showed that the relationship between the two conformed to the Logistic curve model and had a high correlation (r 2 =0.9558), the equation is:

[0065] y = A2 + (A1-A2) / (1 + (x / x0)^p) (1)

[0066] x: whitefly population density,

[0067] y: relative expression level of TYLCV gene.

[0068] pass Figure 2 The equation curve shows that when the population density is 50, 100, and 200, the relative expression of the viral gene does not change significantly; when the density is greater than 400 (148 heads / m 3 ), the viral gene expression began to increase significantly; when the whitefly population density reached 600 (222 / m 3 ), the viral gene expression reached its maximum value; after the population density reached 800 or 1000, the viral gene expression reached saturation and tended to be stable.

[0069] Experiment 2: When measuring the effect of temperature x1 on tomato TY virus infection

[0070] In step S1, a total of 6 temperature gradients of 24°C, 26°C, 28°C, 30°C, 32°C, and 34°C are set in the incubator, a 30×30×30 cm cage is placed in each incubator, and 4 tomato seedlings are placed in each cage;

[0071] In step S2, two diagonal tomato seedlings are used as inoculated plants and inoculated with tomato TY virus;

[0072] In the step S3, one week after the tomato TY virus is inoculated, whiteflies are placed in each cage, and 50 whiteflies are inoculated;

[0073] In the step S4, non-inoculated strains are sampled once every 3 days, for a total of 5 times.

[0074] Tomatoes were inoculated with tomato yellow leaf curl virus (TYLCV) and sampled at 3, 6, 9, and 12 days for qPCR detection and analysis.

[0075] The results showed that at 28°C, the ability of whiteflies to transmit TY virus was stronger. As the temperature rose to 30-34°C, it was not the best temperature for whiteflies to transmit the virus. At this time, the transmission efficiency was lower. Figure 3 shown.

[0076] The peak was reached on the 9th day after inoculation with whiteflies. The correlation between the viral load (Y) and temperature (X) on the 9th day was fitted and a correlation model was established, and the correlation was high (r 2 =0.9667), such as Figure 4 .

[0077] Correlation model between viral load (Y) and temperature (X):

[0078] Y=0.0672x 3 -6.2941x 2 +193.94x-1940.5 (2)

[0079] The expert evaluation method was used to evaluate the weights of temperature and whitefly population density in the occurrence of tomato TY virus disease, and the average values ​​were calculated. The weight of temperature was 0.66, and the weight of population density was 0.34. The weight method was used to model the relationship between temperature, population density and virus load (Equation 1 and Equation 2), and a correlation model was established.

[0080] Y=0.01x1 3 -0.94x1 2 +29.1x1+0.85[A2+(A1-A2) / (1+x2 / x0)]-291.08 (3)

[0081] in,

[0082] Y: relative expression of viral genes;

[0083] x1: temperature;

[0084] x2: population density;

[0085] A1: 2.32684±3.15992;

[0086] A2: 36.98764±3.15992;

[0087] x0:45.01±6.38415.

[0088] The present invention also includes a method for culturing tomato TY virus: using an inoculation loop to dip bacterial liquid from an infectious cloned bacterial strain stored at -70°C, streaking on a YEB solid culture medium, culturing at 28°C to obtain colonies, completing the cultivation of tomato TY virus, and using a sterile toothpick to pick up the colonies and smear them on the back of tomato leaves and tender stems for inoculation.

[0089] YEB solid culture medium contains 50ug / mL kanamycin and 50ug / mL rifampicin.

[0090] Verify the early warning model of formula 3:

[0091] Temperature: 28 degrees, population density: 50, 100, 150, 200, 300 heads / m 3

[0092] 1. Effect of different population densities on TY virus content

[0093] In the incubator, at 28°C, set the temperature to 50 heads / m 3 , 100 heads / m 3 , 150 heads / m 3 , 200 heads / m 3 , 300 heads / m 3 There were 5 population density gradients in total. A 30×30×30 cm cage was placed in each incubator, and 4 tomato seedlings were placed in each cage. Two diagonal tomato plants were inoculated with TY virus, and one week after the inoculation with TY virus, they were inoculated with whiteflies. After the inoculation with whiteflies, samples were taken every 3 days to detect the virus load in the plants that were not inoculated with the virus.

[0094] As shown in the survey results in Table 1, the actual measured values ​​of TY virus content under different population density treatments were basically consistent with the theoretical values, indicating that the prediction model was in line with expectations.

[0095] Table 1 Relative load of TY virus in tomato plants under different population density treatments

[0096]

[0097] 2. Determination of TY virus content at different temperatures

[0098] The incubator was set with 4 temperature gradients of 24℃, 26℃, 28℃, 30℃, and 32℃. A 30×30×30cm cage was placed in each incubator, and 4 tomato seedlings were placed in each cage. Two diagonal tomato plants were inoculated with TY virus, and one week after the inoculation with TY virus, whiteflies were inoculated, and each cage was inoculated with 200 whiteflies / m3. Samples were taken every 3 days after the inoculation of whiteflies to detect the virus load in the plants that were not inoculated with the virus.

[0099] The survey results show that under different temperature treatments, the actual measured values ​​of the virus content are basically consistent with the theoretical values, indicating that the prediction model is in line with expectations.

[0100] Table 2 Relative load of TY virus in tomato plants under different temperature treatments

[0101]

[0102] Before the experiment, TYLCV was detected on tomato seedlings and whiteflies. The detection method was as follows: genomic DNA was extracted from tomato seedling leaves and whiteflies, TYLCV-specific gene primers TYLCV-61 and TYLCV-473 were used for PCR amplification, and the obtained PCR products were detected by 1.2% agarose gel electrophoresis.

[0103] The reaction procedure of the PCR amplification was: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, and extension at 68°C for 1 s, for a total of 35 cycles.

[0104] The sequence of the TYLCV-specific gene primer TYLCV-61 is "5'-ATACTTGGACACCTAATGGC-3'", and the sequence of TYLCV-473 is "5'-AGTCACGGGCCCTTACAA-3'".

[0105] Instructions attached Figure 5 In the figure, Y1-3 is a tomato leaf; F1-3 is a whitefly; TY1-4 is an Agrobacterium containing the TY virus. Through PCR amplification, the tomato whitefly cultured in the laboratory was tested for the presence of the Tomato Yellow Leaf Curl Virus (TYLCV). The results showed that the Agrobacterium containing TYLCV could amplify a 412bp virus-specific gene fragment, while the laboratory-cultivated tomatoes and the reared whitefly populations did not amplify the virus-specific gene fragment, indicating that the laboratory-cultivated tomatoes and the reared whitefly populations themselves did not carry the Tomato Yellow Leaf Curl Virus, which met the experimental requirements.

Claims

1. A method for early warning of tomato TY virus occurrence based on temperature and whitefly population density, characterized by: A correlation model among the relative expression of viral genes Y, temperature x1 and whitefly population density x2 was established, and the temperature x1 and whitefly population density x2 in the tomato planting field were measured. When the relative expression of viral genes Y obtained through the correlation model exceeded the warning value, a prevention and control alarm was triggered. The correlation model is: <h2 style=";text-align:left;direction:ltr">Y = 0.01x1<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> -0.94x1<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> +29.1x1+0.85[A2+(A1-A2) / (1+x2 / x0)]-291.08 in, Y: relative expression of viral genes; x1: temperature; x2: population density; A1：2.32684±3.15992； A2：36.98764±3.15992； x0：45.01±6.38415。 2. The early warning method according to claim 1, characterized in that: The method for establishing the correlation model comprises the following steps: S1. Build cages and cover with insect-proof nets, and plant several tomato seedlings in each cage; S2. After planting, select inoculated and non-inoculated strains in the cage, and inoculate the inoculated strains with tomato TY virus; S3. After inoculation, whiteflies are placed in the cage according to the set population density; Sampling was started on the third day after the release of whiteflies to detect the viral load in the plants not inoculated with the virus. The relative expression level Y of the viral gene in the leaves was analyzed by qPCR, and a correlation model was established based on the greenhouse temperature x1 and the whitefly population density x2 of the corresponding treatment.

3. The early warning method according to claim 2, characterized in that: When the effect of whitefly population density x2 on tomato TY virus infection was measured, In the step S1, eight 1.2×1.5×1.5 meter cages are built in the greenhouse, covered with 60-mesh insect-proof nets, and 12 tomato seedlings are planted in each net cover, arranged in a 4×3 pattern to form a seedling array; In the step S2, 10 days after the seedlings are planted, two tomato seedlings in the middle of each seedling array in each cage are used as inoculated plants and inoculated with tomato TY virus; In the step S3, whiteflies were placed one week after inoculation of tomato TY virus. The experiment had 7 treatments and 1 control. 0, 50, 100, 200, 400, 600, 800, and 1000 whiteflies were placed respectively, of which 0 was the control group. In step S4, 5 plants are randomly selected from the 10 non-inoculated plants at the inner edge of each cage for sampling, as 5 repetitions, and the top leaves are picked for sampling, and the sampling is carried out once every 3 days, for a total of 4 times.

4. The early warning method according to claim 2, characterized in that: When measuring the effect of temperature x1 on tomato TY virus infection, In step S1, a total of 6 temperature gradients of 24°C, 26°C, 28°C, 30°C, 32°C, and 34°C are set in the incubator, a 30×30×30 cm cage is placed in each incubator, and 4 tomato seedlings are placed in each cage; In step S2, two diagonal tomato seedlings are used as inoculated plants and inoculated with tomato TY virus; In the step S3, one week after the tomato TY virus is inoculated, whiteflies are placed in each cage, and 50 whiteflies are inoculated; In the step S4, non-inoculated strains are sampled once every 3 days, for a total of 5 times.

5. The early warning method according to claim 1, characterized in that: It also includes a method for culturing tomato TY virus: use an inoculation loop to dip the bacterial liquid from the infectious clone strain preserved at -70°C, streak on YEB solid culture medium, culture at 28°C to obtain colonies, complete the cultivation of tomato TY virus, and use a sterile toothpick to pick up the colonies and smear them on the back of tomato leaves and tender stems for inoculation.

6. The early warning method according to claim 5, characterized in that: YEB solid culture medium contains 50ug / mL kanamycin and 50ug / mL rifampicin.

7. The early warning method according to claim 2, characterized in that: Before the experiment, tomato seedlings and whiteflies were tested for TYLCV. The detection method was as follows: genomic DNA of tomato seedling leaves and whiteflies was extracted, PCR amplification was performed using TYLCV-specific gene primers TYLCV-61 and TYLCV-473, and the obtained PCR products were detected by 1.2% agarose gel electrophoresis. If no virus-specific gene fragments were amplified, it indicated that the laboratory-cultivated tomatoes and the reared whitefly populations themselves did not carry tomato yellow leaf curl virus, and met the experimental requirements.

8. The early warning method according to claim 7, characterized in that: The reaction procedure of the PCR amplification was: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, and extension at 68°C for 1 s, for a total of 35 cycles.

9. The early warning method according to claim 7, characterized in that: The sequence of the TYLCV-specific gene primer TYLCV-61 is "5'-ATACTTGGACACCTAATGGC-3'", and the sequence of TYLCV-473 is "5'-AGTCACGGGCCCTTACAA-3'".

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