CN103404397A - Method for preventing and treating candidatus liberibacter asiaticus by heat treatment - Google Patents

Abstract

The invention specifically discloses a method for preventing and treating citrus huanglongbing by using heat treatment. The specific steps are to place the citrus trees infected with Huanglongbing in an environment of 45-52°C for continuous heat treatment for 2-5 hours, and then place the trees at ambient temperature for cultivation; repeat the above operation in the next week, and continue the treatment for 3-4 hours. week. The method used in the invention is economical and environment-friendly, can greatly reduce or eliminate the content of pathogenic bacteria in the diseased trees, restore the vigor of the experimental trees and grow normally, and significantly improve the yield and quality of citrus.

Description

A method of preventing and treating citrus huanglongbing by heat treatment

technical field

The invention belongs to the technical field of plant protection, and in particular relates to a method for preventing and treating citrus huanglongbing by heat treatment.

Background technique

Citrus Huanglongbing is the most serious and devastating disease in citrus production. The disease was first discovered in Chaoshan area of Guangdong, my country in 1919, and has occurred and harmed more than 50 countries and regions so far. Among the 17 citrus producing areas in my country, 11 are under the threat of Huanglongbing, and Guangdong, Guangxi, Fujian, Yunnan, and Hainan, which are the main citrus producing areas, are particularly serious. Typical symptoms of affected citrus plants are "yellow shoots", mottled and yellowed leaves, smaller fruit, and the formation of "green fruit" or "red nose fruit" with no economic value. In the late stage of the disease, the root system is necrotic, and the tree vigor declines, resulting in leaf drop, fruit drop, and plant death.

The causative agent of citrus greening disease is the bacillus " Candidatus Liberibacter spp" confined to the phloem, which is transmitted by grafting and feeding by psyllids. " Ca . L. spp" is divided into the heat-tolerant Asian species (" Ca . L. asiaticus") that is currently the dominant population, the temperature-sensitive American species (" Ca . L. americanus") that was once prevalent in Brazil, and the African species ("Ca. L. africanus") endemic to South Africa. The pathogen can be detected in bark tissue, leaves, flowers, roots and fruits.

For Huanglongbing, which has been plagued by the epidemic for hundreds of years, there is currently no feasible method that can effectively directly reduce or eliminate the pathogen in the diseased plants. There is a lack of seed capital resources for resistance/resistance to Huanglongbing in citrus varieties, so people have adopted integrated control methods to control the disease for more than 100 years: first, check the seedlings, strengthen quarantine, and select disease-free seedlings and scions; Strengthen cultivation and management of mildly diseased trees (reasonable fertilization and irrigation, removal of summer buds, pruning of elongated branches, appropriate fruit thinning, etc.), timely pruning of diseased branches, and injection of antibiotics; In the way of transmission, chemical, physical and biological methods are used to control citrus psyllids. However, the use of disease-free seedlings cannot avoid infection and spread in the field; injection of antibiotics and chemical control methods not only pollute the environment but also affect food safety; methods of digging or cutting down diseased trees will directly block citrus production. China and India used hot water to treat diseased buds and diseased scions as early as the 1960s and 1970s, and the grafted citrus obtained a good detoxification effect. But there is no report on the heat treatment of Huanglongbing trees at the adult stage.

The characteristics of the patent "Using Biological Factors to Control the Spread of Citrus Huanglongbing Disease" emphasizes the use of insecticides to clear the garden in winter and spray insecticides and acaricides before releasing mites; Psyllids control re-infestation, ie spread, of Huanglongbing and have no therapeutic effect on already infected plants.

Contents of the invention

The object of the present invention is to provide a method for preventing and treating citrus huanglongbing by heat treatment in order to solve the defect that the prevention and control technology of citrus huanglongbing is not effective in the prior art.

The present invention realizes above-mentioned object through following technical scheme:

A method for preventing and treating citrus huanglongbing by heat treatment, comprising the following steps: placing citrus trees infected with huanglongbing in an environment of 45-52° C. for continuous heat treatment for 2-5 hours, and then cultivating the trees at ambient temperature; Repeat the above operation in the following week, and continue to treat for 3 to 4 weeks.

The citrus trees infected with huanglongbing are placed in an environment of 45-52°C for 2-5 hours of continuous heat treatment. This treatment can be determined according to the cultivation environment of citrus trees. Measures to provide an environment of 45-52°C. No matter what kind of measures, as long as the infected citrus tree body can be continuously heat-treated for 2-5 hours under the environment of 45-52 ℃.

The citrus trees infected with Huanglongbing include field citrus trees infected with Huanglongbing and potted citrus seedlings infected with Huanglongbing.

For the diseased citrus trees in the field, the following methods can be used to carry out heat treatment, and the specific steps are as follows:

S1. Build a frame: build a frame around the citrus trees in Daejeon that have been infected with Huanglongbing;

S2. Covering with plastic film: Choose a sunny day with the highest daily temperature greater than 35°C, completely cover the periphery of the frame with plastic film, so that the internal temperature of the film reaches 45-52°C, and place the citrus tree in an environment of 45-52°C for continuous After 2 to 5 hours of heat treatment, the film is uncovered, and the tree body is cultivated at ambient temperature; the above operation is repeated for the next week, and the treatment is continued for 3 to 4 weeks. Of course, the Daejeon citrus tree can also adopt other methods to provide an environment of 45-52°C.

For the potted citrus seedlings infected with the disease, heat treatment can be carried out in the following way, the specific steps are as follows: put the potted citrus seedlings infected with Huanglongbing in a temperature control box at 45-52°C, continue heat treatment for 2-5 hours, and then remove the potted citrus seedlings from Take out the temperature control box and place it at ambient temperature for cultivation; repeat the above operation in the next week, and continue the treatment for 3 to 4 weeks.

Or you can use the following methods, the specific steps are:

S1. Build a frame: build a frame around the citrus potted seedlings infected with Huanglongbing;

S2. Covering with plastic film: Choose a sunny day with the highest daily temperature greater than 35°C, completely cover the periphery of the frame with plastic film, so that the internal temperature of the film reaches 45-52°C, and place the citrus tree in an environment of 45-52°C for continuous After 2 to 5 hours of heat treatment, the film is uncovered, and the tree body is cultivated at ambient temperature; the above operation is repeated for the next week, and the treatment is continued for 3 to 4 weeks. Of course, other ways can also be used to provide an environment of 45-52°C for potted seedlings of infected citrus. As long as the measure that can provide 45～52 ℃ environment for the infected citrus tree body is applicable to the present invention.

The material used to build the frame can be selected from dry bamboo; it can also be made of hollow iron pipes or stainless steel pipes connected in series; the frame built by the former can be reused at fixed points, while the latter has strong mobility and can be recycled for many years.

The plastic film described in step S2 has a thickness of 6s and a width of 6m. The required plastic film for large trees is wider, and can be glued with a wide 4.4 to 5.5 cm scotch tape to ensure that the plastic film can completely cover the tree body and not be close to the leaves.

In terms of temperature control, the plastic film should be covered between 11:00 noon and 5:00 pm on a sunny day with the highest temperature greater than 35°C in summer and autumn, and electronic temperature recorders should be placed on the upper, middle, and lower parts of the tree.

A small amount of damaged leaves and branches should be pruned after each heat treatment.

Placing the infected citrus trees at 45-52°C for 2-5 hours of continuous heat treatment will not cause harm to the citrus trees. Various experiments have proved that high temperature will cause damage to a few branches and leaves that are close to the film, so later pruning is required . If the film is not attached to the tree, there will be no damage: first, it has the self-regulation ability of the tree; second, the temperature of the root is lower than the ambient temperature, and the root is the key to plant growth; third, citrus is a woody plant , its organizational structure determines that it is less sensitive to temperature than ordinary herbs.

The present invention research finds: the high-temperature treatment method of the present invention has no difference for the effect between the citrus trees of different varieties and different morbidity levels, but in terms of tree age, the heat treatment time of potted seedlings should be shorter, 2 ~ 3 The treatment time for adult trees in the field should be longer, preferably 4 to 5 hours.

The invention uses an economical and environment-friendly heat treatment method to kill pathogenic bacteria in the tree without using chemical agents, and focuses on the purpose of facilitating the restoration of citrus tree vigor and increasing the yield, and constructs a physical control centered on the combination of cultivation management, natural regulation and pest control. A green prevention and control system for prevention and control.

Compared with the prior art, the present invention has the following beneficial effects:

1. The energy used in the present invention is natural solar energy, without using chemical substances such as antibiotics, pesticides, fertilizers, etc., which is very economical and environmentally friendly. The scaffolding constructed can be used repeatedly, and can be used as an auxiliary tool for orchard farming operations such as wiping, supporting branches and picking.

2. It is proved by experiments that the concentration of the pathogen " Ca. Liberibacter asiaticus" in the citrus Huanglongbing tree after heat treatment by the method of the present invention is significantly reduced or even eliminated, the tree body grows vigorously, and the resulting fruit quality recovers Normal, significantly higher yields.

Instructions attached

Figure 1. CT value (A) and copy number of “ Ca. L. asisticus” (B ) changes; columns marked with different letters indicate significant differences.

Fig. 2. Changes in the copy number of " Ca. L. asisticus" of 12 Huanglongbing trees in the 8-year-old sweet orange treatment group and the control group in Huizhou, Guangdong, before treatment, 30 days, 60 days and 90 days after treatment; "T" indicates treated plants and "C" indicates control plants.

Fig. 3. " Ca . L" in the leaves of 6-year-old Shatangju treated with heat treatment in Qingyuan, Guangdong, and 13 Shatangju infected with Huanglongbing in the control group before treatment and 30 days, 60 days, 90 days and 150 days after treatment .asisticus" copy number change; where "T" indicates treated plants and "C" indicates control plants.

Fig. 4. Symptoms of 7-year-old Shatangju plants in Qingyuan, Guangdong before and after heat treatment; where A, B and C are before treatment, and A’, B’ and C’ are 2 months after treatment.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

Example 1

There is a 200-square-meter net room in Changgang Mountain on the campus of South China Agricultural University.

Select citrus trees of various varieties, ages and disease degrees that have been infected with Huanglongbing as treatment objects. The processing object of this embodiment is to select 18 3-year-old infected tangerine potted seedlings in the autumn of 2011, and adopt the method of net room plastic film heat treatment to carry out heat treatment to the infected citrus potted seedlings. The specific steps are as follows:

S1. Build a frame: build a frame around the citrus potted seedlings infected with Huanglongbing;

S2. Covering with plastic film: Choose a sunny day with the highest daily temperature greater than 35°C, completely cover the periphery of the frame with plastic film, so that the internal temperature of the film reaches 45-52°C, and place the citrus tree in an environment of 45-52°C for continuous After 2 to 5 hours of heat treatment, the film is uncovered, and the tree body is cultivated at ambient temperature; the above operation is repeated for the next week, and the treatment is continued for 3 to 4 weeks.

Samples were collected 30 days and 90 days after the treatment, and the pathogenic content of Huanglongbing in the samples was detected by the method of Real time PCR. The results showed that there was no significant difference in the CT value between the treatment group and the control group before treatment (P>0.05), but the CT value of the two groups increased significantly (that is, the pathogen concentration was extremely significantly reduced) at 30 days and 90 days after treatment (P<0.01) ) (see Table 1 for the results). It shows that heat treatment of plastic film has a good effect on inhibiting " Ca . L. asisticus" in potted citrus seedlings.

Table 1 The change of CT value of “ Ca. L. asisticus” in potted citrus seedlings treated with plastic film in net house with time

Note: There are significant differences between those with different letters in the same column (DMRT method)

Example 2

In the spring of 2012, 24 3-year-old potted seedlings of Shatangju were selected for heat treatment in a temperature control box. The specific heat treatment method was as follows: the 24 seedlings were randomly assigned to 4 treatments: 42°C, 46°C, 50°C And control, wherein each temperature is treated once a week, every time 2 hours, and is treated 4 times in a row, that is, 4 weeks of continuous treatment. After the high temperature treatment, the potted seedlings are taken out from the temperature control box and placed at ambient temperature for cultivation.

Samples were collected 30 days, 60 days and 150 days after the treatment, and Real time PCR was used to detect the pathogenic content of Huanglongbing in the samples. The results showed that there was no significant difference in the CT values of the control group and the 42°C treatment group before and after several months of treatment (P>0.05); while the CT values of the 46°C and 50°C treatment groups were 30 days, 60 days, and 150 days after treatment Compared with the 42°C treatment group and the control group, it was significantly increased (that is, the pathogen concentration was significantly reduced) (P<0.05); the difference between the 46°C and 50°C treatment groups was not significant (P>0.05) (Table 2). The results showed that treatment at 42°C for 8 h could not significantly inhibit " Ca . L. asisticus", while treatment at 46°C-50°C for 8 h could significantly reduce the concentration of " Ca . L. asisticus".

Table 2 Changes of CT values over time in post-emergence samples of potted Huanglongbing disease treated with temperature control box (DMRT method)

Note: There are significant differences between those with different letters in the same column (DMRT method)

Example 3

Yangcun, Boluo County, Huizhou City, Guangdong Province is the hardest-hit area for citrus Huanglongbing. The Yangcun Citrus Farm established in 1951 is one of the famous citrus fields in the country. By the early 1970s, its scale reached 20,000 mu (1 million plants) tree), in 1972 the overall profit reached 1.45 million yuan. However, since 1974 Huanglongbing became popular, and it occurred seriously from 1977 to 1982, which eventually led to the destruction of large areas, and the sporadic citrus orchards began to disperse to self-employed individuals. In 2011, our laboratory cooperated with Guan Meiying, a fruit farmer in Yangcun, to carry out heat treatment experiments on the basis of ensuring the normal management of the orchard. Select 24 strains of 8-year-old sweet orange tree bodies that are severely morbid as experimental materials, and process 12 of them with the method of plastic film covering heat treatment; the method concrete steps of plastic film covering heat treatment are:

S1. Build the frame: build a frame around the diseased tree according to the size of the tree; the material used to build the frame can be made of dried bamboo; it can also be made of hollow iron pipes or stainless steel pipes connected in series; the frame built by the former can be fixed-point Repeated use, the latter has strong mobility and can be recycled for many years.

S2. Covering with plastic film: choose a sunny day with the highest daily temperature greater than 35°C, and cover the outside of the frame with an ordinary plastic film completely, so that the temperature inside the film reaches 45-52°C and lasts for 5 hours; uncover the film after treatment , Repeat the above operation every other week, a total of 3 times a year. The thickness of the plastic film is 6s, and the width is 6m. For large trees, the required plastic film is wider, and it can be glued with transparent tape with a width of 4.4-5.5 cm to ensure that the plastic film can completely cover the tree body and not cling to the leaves. In terms of temperature control, the plastic film should be covered between 11:00 noon and 5:00 pm on sunny days with the highest temperature greater than 35°C in summer and autumn, and electronic temperature recorders should be placed on the upper, middle, and lower parts of the tree. A small amount of damaged leaves and branches should be pruned after each treatment.

The invention uses an economical and environment-friendly heat treatment method to kill pathogenic bacteria in the tree without using chemical agents, and focuses on the purpose of facilitating the restoration of citrus tree vigor and increasing the yield, and constructs a physical control centered on the combination of cultivation management, natural regulation and pest control. A green prevention and control system for prevention and control.

Before the treatment, 1 month, 2 months and 3 months after the treatment, the real time PCR method was used to detect the content of the pathogen " Ca. L. asisticus" in the trees. The results showed that the CT values at 30 days, 60 days and 90 days after treatment were significantly higher than those before treatment (P<0.001); the corresponding " Ca . % and 85.2% (see Figure 1 for the results). The copy number of " Ca. L. asisticus" in all treatment trees decreased at 30 days and 90 days after treatment, while the change pattern of the copy number of " Ca. L. asisticus" in the control group was inconsistent (see Figure 2 for results). This experiment shows that heat treatment of plastic film plays a good role in inhibiting " Ca . L. asisticus".

Example 4

Since 1996, Xinda Farm in Taiping Town, Qingxin County, Qingyuan City, Guangdong Province has planted 285 mu of Tangerine. In the winter of 2011, my laboratory conducted a comprehensive investigation on the occurrence of Huanglongbing disease on the farm, and found that the incidence rate of Huanglongbing disease was as high as 24.9%. In July 2012, we selected 26 7-year-old trees with yellow dragon disease in the orchard to conduct plastic film heat treatment experiments (13 diseased trees were used as treatments, and the other 13 were used as controls). The specific steps of the plastic film covering heat treatment method were the same as the implementation Example 3.

Before the treatment, 30 days, 60 days, 90 days, 150 days and 210 days after the treatment, the real time PCR method was used to detect the pathogenic content of Huanglongbing in the leaves and roots of the diseased trees. The results found that: 30 days, 60 days after the treatment The CT value of day, 90 day, 150 day and 210 day was significantly higher than the level before treatment (P<0.0001), and the concentration of " Ca. L. asisticus" in the treatment group was 8.2, 1.31, 7.11, 0.39 and 0.12 fg/μL, an average decrease of 99.03%, 99.84%, 99.15%, 99.98% and 99.99% compared with before treatment. From an individual point of view, the concentration of " Ca. L. asisticus" in the 13 trees in the treatment group was reduced by more than 95% in several months after treatment; Compared with before the experiment, there was no significant difference (P>0.05), and the average copy number of " Ca . L. asisticus" increased by 21.72%, 5.52%, 120% and decreased by 0.8% respectively at the corresponding time compared with before the experiment (result See Figure 3). This experiment shows that the method of heat treatment covered with plastic film has a significant effect on the control of citrus Huanglongbing in the field: significantly reducing the pathogen content in the tree body and significantly reducing symptoms and improving tree vigor (results shown in Figure 4).

The present invention research finds: the high-temperature treatment method of the present invention has no difference for the effect between the citrus trees of different varieties and different morbidity levels, but in terms of tree age, the heat treatment time of potted seedlings should be shorter, 2 ~ 3 The treatment time for adult trees in the field should be longer, preferably 4 to 5 hours.

Claims (7)

1. a method of utilizing thermal treatment control Citrus Huanglongbing pathogen, is characterized in that, comprises the steps: that the mandarin tree body that will dye yellow twig is placed under 45～52 ℃ of environment to continue thermal treatment 2～5 hours, then will set body and be placed under environmental temperature and cultivate; Next a week repeats above operation, processes continuously for 3～4 weeks.

2. method according to claim 1, is characterized in that, the described mandarin tree body that has dyed yellow twig comprises the land for growing field crops mandarin tree body that dyes yellow twig and the potted plant seedling of oranges and tangerines that has dyed yellow twig.

3. method according to claim 1, is characterized in that, comprises the steps:

S1. build framework: build framework in the mandarin tree body periphery, land for growing field crops of dying yellow twig;

S2. plastic covering film: the fine day of selecting daily maximum temperature to be greater than 35 ℃, the plastic covering film intactly in the periphery of building framework, make the film internal temperature reach 45～52 ℃, the mandarin tree body is placed under 45～52 ℃ of environment and continues thermal treatment 2～5 and open film after little, will set body and be placed under environmental temperature and cultivate; Next a week repeats above operation, processes continuously for 3～4 weeks.

4. method according to claim 1, it is characterized in that, comprise the steps: the potted plant seedling of the oranges and tangerines that dye yellow twig is placed in the temperature control box of 45～52 ℃, continue thermal treatment 2～5 hours, then the potted plant seedling of oranges and tangerines is taken out to be placed under environmental temperature from temperature control box and cultivate; Next a week repeats above operation, processes continuously for 3～4 weeks;

Perhaps comprise the steps:

S1. build framework: build framework in the potted plant seedling of the oranges and tangerines that dye yellow twig periphery;

S2. plastic covering film: the fine day of selecting daily maximum temperature to be greater than 35 ℃, the plastic covering film intactly in the periphery of building framework, make the film internal temperature reach 45～52 ℃, the mandarin tree body is placed under 45～52 ℃ of environment and continues thermal treatment 2～5 and open film after little, will set body and be placed under environmental temperature and cultivate; Next a week repeats above operation, processes continuously for 3～4 weeks.

5. method according to claim 3, is characterized in that, the described material of building framework of step S1 is dry bamboo, hollow iron pipe or stainless steel tube.

6. according to the described method of claim 3 or 4, it is characterized in that, described plastic foil thickness is 6s, and width is 6m.

7. according to the described method of claim 1 to 4 any one, it is characterized in that, blade and the branch of a small amount of damage of each thermal treatment reply are pruned.

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