Selection and breeding methods based on the evaluation model of citrus Huanglongbing

Abstract

The present invention relates to the field of model judgment breeding, and in particular to a breeding method and a breeding method based on a citrus Huanglongbing evaluation model. The present invention finds a judgment model capable of combining and judging whether citrus is infected with Huanglongbing based on the previous research on Huanglongbing metabolomics and many years of field test observation experience, namely: when the nitrogen-green ratio is ≤0.150 and the sugar-green ratio is ≥0.10, the plant is infected with Huanglongbing; when the nitrogen-green ratio is >0.150 and / or the sugar-green ratio is <0.10, the plant is not infected with Huanglongbing; and the conclusion of the model is consistent with the verification result of PCR. The construction of this model can simply and quickly detect data and perform analysis and judgment only through a handheld chlorophyll meter and a handheld plimeter, thus providing a reliable and convenient method for the rapid initial judgment of Huanglongbing. Citrus varieties and / or strains with nitrogen-green ratios >0.150 and sugar-green ratios <0.10 selected by using the model can be used as varieties and / or strains with high resistance to Huanglongbing.

Description

[Technical field]

[0001] The present invention relates to the field of model-based breeding, and in particular to a breeding method and a breeding method based on a citrus Huanglongbing evaluation model. [Background technology]

[0002] Huanglongbing is one of the most serious and destructive diseases in the world that harms citrus production. Citrus Huanglongbing is caused by a Gram-negative bacterium that is confined to the phloem and can infect a variety of Rutaceae plants including Citrus, Trifoliate Orange, Fortunella and Murraya. It is a devastating disease in citrus production around the world. The pathogen is a Gram-negative bacterium belonging to the genus Bacillus. Most of the bacteria are round or oval, and a few are irregular in shape. The size is (20-600) nanometers × (170-1600) nanometers, 17-33 nanometers thick, 25 nanometers on average, and composed of three layers of membranes.

[0003] At present, there are two most commonly used methods for citrus Huanglongbing in the prior art, one is the method of direct observation and disease grading, and the other is the method of using PCR for detection; although the first method has low cost, it requires the observer to have high experience, which is not friendly to novice fruit farmers, and the observer often makes misjudgments, because the phenotype of Huanglongbing is very similar to other citrus diseases, nutrient deficiencies and other symptoms, and direct observation requires a period of time after infection with the Huanglongbing pathogen to be reflected in the phenotype, which will delay the prevention period; for the second method, using PCR to directly detect the nucleic acid of the pathogen is more accurate and precise, and in the early stage of the disease, when the citrus does not show phenotypic symptoms, as long as the pathogen reaches a certain amount, it can be detected by PCR, but this method requires a PCR detector, and for the operator, it requires high experimental skills and instrument analysis level, and is not suitable for fruit farmers to carry out detection in the field.

[0004] In addition to the two conventional methods mentioned above, the current prior art discloses: 202210097151.8 "A kit and method for rapid detection of citrus Huanglongbing based on colorimetry" also describes a method for determining whether citrus is infected with Huanglongbing by measuring starch. This method is based on previous metabolomics research on citrus Huanglongbing. The results show that the accumulated starch content in the leaves of plants infected with citrus Huanglongbing is greater than that in uninfected leaves. Therefore, the color development of starch iodine solution can be used to determine whether citrus is infected with Huanglongbing. This method has been made into a kit for sale. However, we found in the process of use that the color development is deep and shallow, and the person making the judgment is relatively subjective, and sometimes the judgment of the depth of color will be misjudged.

[0005] In the leaf metabolomics study of citrus Huanglongbing, it was found that infection with citrus Huanglongbing often affects the starch metabolism pathway, amino acid metabolism pathway, fat metabolism pathway and sugar metabolism pathway of the leaves. Therefore, suitable markers can be selected according to the above-mentioned metabolic pathways. However, since the detection of most markers requires the operator to have high experimental operation capabilities and requires high and expensive equipment, how to achieve a relatively accurate preliminary judgment of citrus Huanglongbing through simple methods is a technical problem that urgently needs to be solved. [Summary of the invention]

[0006] In view of the above, it is necessary to conduct a comprehensive and unified analysis of various evaluation indicators to derive a new evaluation method and mathematical model that can evaluate the cold resistance characteristics of sugarcane simply and efficiently.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for quickly determining the susceptibility of citrus Huanglongbing based on a citrus Huanglongbing evaluation model, the method comprising: randomly taking leaves from citrus plants, using a handheld chlorophyll meter and a handheld plummeter to measure the SPAD value, nitrogen content and sucrose content of the leaves, and then calculating the nitrogen-green ratio and the sugar-green ratio; when the nitrogen-green ratio is ≤0.150 and the sugar-green ratio is ≥0.10, the plant is infected with Huanglongbing; when the nitrogen-green ratio is >0.150 and / or the sugar-green ratio is <0.10, the plant is not infected with Huanglongbing.

[0009] A method for breeding highly resistant Huanglongbing varieties and / or strains based on a citrus Huanglongbing evaluation model, the method comprising: randomly picking leaves from citrus plants in a Huanglongbing disease area, measuring the SPAD value, nitrogen content and sucrose content of the leaves with a handheld chlorophyll meter and a handheld plummeter, and then calculating the nitrogen-to-green ratio and the sugar-to-green ratio, and breeding citrus varieties and / or strains with a nitrogen-to-green ratio>0.150 and a sugar-to-green ratio<0.10.

[0010] A method for breeding highly resistant Huanglongbing varieties and / or strains based on a citrus Huanglongbing evaluation model, the method comprising: randomly taking leaves from citrus plants, measuring the SPAD value, nitrogen content and sucrose content of the leaves with a handheld chlorophyll meter and a handheld plummeter, then calculating the nitrogen-to-green ratio and the sugar-to-green ratio, selecting citrus plant branches with a nitrogen-to-green ratio>0.150 and a sugar-to-green ratio<0.10 as scions, and performing grafting or cross-breeding on the citrus.

[0011] Furthermore, the leaf sampling method is: take 5 different branches from each tree, take 8 leaves from each branch, take 3 leaves from the top leaves of the new shoots, and take 5 leaves from each of the 3rd layer from the top.

[0012] Furthermore, the nitrogen-green ratio is calculated as follows: leaf nitrogen content / SPAD value, and the sugar-green ratio is calculated as follows: leaf brix / SPAD value.

[0013] Furthermore, the grafted rootstock is Fructus Aurantii Immaturus.

[0014] The present invention has the following beneficial effects:

[0015] 1. The present invention has found a judgment model that can be combined to judge whether citrus is infected with Huanglongbing based on the previous research on Huanglongbing metabolomics and many years of field test observation experience. In many years of field and online monitoring, we found that the leaf nitrogen content / SPAD value of citrus plants infected with Huanglongbing was significantly lower than that of non-infected plants under the same greenness conditions; the leaf brix / SPAD value was significantly higher than that of non-infected plants under the same greenness conditions; based on this, through a large amount of data verification, collection and model adjustment, we found that: when the nitrogen-green ratio is ≤0.150 and the sugar-green ratio is ≥0.10, the plant is infected with Huanglongbing; when the nitrogen-green ratio is >0.150 and / or the sugar-green ratio is <0.10, the plant is not infected with Huanglongbing; after PCR detection and verification, we found that the conclusion is consistent with the verification result of PCR. The construction of this model can simply and quickly detect data and perform analysis and judgment only through a handheld chlorophyll meter and a handheld brix meter, which provides a reliable and convenient method for the rapid initial judgment of Huanglongbing.

[0016] 2. The present invention, through grafting breeding, has obtained a method for breeding plants with high resistance to Huanglongbing on the basis of the citrus Huanglongbing evaluation model, namely: citrus varieties and / or strains with nitrogen-green ratio>0.150 and sugar-green ratio<0.10 are selected as plant parents or scions, which provides a quick and convenient method for the next step of breeding plants with high resistance to Huanglongbing. [Specific implementation method]

[0017] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation of the present invention is described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0018] Embodiment 1:

[0019] This example is the index setting process for citrus high resistance to Huanglongbing cold-resistant breeding:

[0020] 1. Determination of Huanglongbing detection model:

[0021] In the previous research and extensive reference, we found that the leaves of citrus plants infected with Huanglongbing were more seriously deficient in nutrients than those simply deficient in nutrients. In addition, sugar metabolism increased, and the sugar content in the leaves was significantly higher than that of plants with the same chlorophyll content. Therefore, based on long-term data investigation, follow-up research and optimization, we have developed a set of methods that do not use PCR methods, but only use handheld chlorophyll meters and handheld pliers through calculation optimization to preliminarily determine whether citrus is infected with Huanglongbing. Moreover, this method can be well applied to the breeding of citrus Huanglongbing. The specific method is: different leaves are selected in the Huanglongbing disease area for field testing, mainly using a handheld chlorophyll meter to detect the SPAD value and total nitrogen content of the leaves; then the corresponding leaves of each plant are collected and ground, half of which is used to measure the sugar content of the leaves with a pliers meter, and the other half of the ground leaf tissue is used for PCR testing according to the PCR determination method to determine whether it is positive for Huanglongbing.

[0022] Among them, the leaf sampling method is: take 5 different branches from each tree, take 8 leaves from each branch, take 3 leaves from the top leaves of the new shoots, take 5 leaves from the third layer from the top, and take the average as the final measured value.

[0023] Then the nitrogen-green ratio (N / SP) and sugar-green ratio (T / SP) are calculated respectively, where:

[0024] Nitrogen-green ratio (N / SP) = leaf nitrogen content / SPAD value; wherein, the unit of leaf nitrogen content is mg / g, that is, if the leaf nitrogen content is 12 mg / g, then 12 is substituted into the formula.

[0025] Sugar-green ratio (T / SP) = blade brix / SPAD value; the unit of blade brix is ​​Br°.

[0026] Then determine the nitrogen-to-green ratio (N / SP) and sugar-to-green ratio (T / SP);

[0027] When the nitrogen-green ratio (N / SP) is ≤0.150 and the sugar-green ratio (T / SP) is ≥0.10, the plant is infected with Huanglongbing;

[0028] When the nitrogen-green ratio (N / SP)>0.150 and / or the sugar-green ratio (T / SP)<0.10, the plant is not infected with Huanglongbing. The specific survey results are as follows:

[0029] Table 1 Results of citrus Huanglongbing disease determination

[0030]

[0031]

[0032] According to Table 1, only plants GJ-2, GJ-5, GJ-6, GJ-8, GJ-9, and GJ-10 with nitrogen-green ratio (N / SP) ≤ 0.150 and sugar-green ratio (T / SP) ≥ 0.10 were detected positive by PCR, and when nitrogen-green ratio (N / SP)> 0.150 or sugar-green ratio (T / SP)< 0.10, although the plants showed adverse phenotypes such as yellow leaves due to other reasons, such as infection with other diseases or insufficient absorption of soil nutrients, they were still not infected with Huanglongbing, indicating that the judgment model of this application is reliable, and a handheld chlorophyll meter and a pliometer can be used to conduct preliminary detection of whether citrus is infected with Huanglongbing. The test results are reliable.

[0033] Embodiment 2:

[0034] This example uses the method of Example 1 to carry out grafting breeding on citrus, specifically as follows:

[0035] The scion is collected from the branches of the virus-free mother plant selected in Table 1 of Example 1. The branches include: sub-healthy branches with nitrogen-green ratio (N / SP)>0.150 or sugar-green ratio (T / SP)<0.10 that do not contain Huanglongbing pathogens and healthy branches with nitrogen-green ratio (N / SP)>0.150 and sugar-green ratio (T / SP)<0.10 that do not contain Huanglongbing pathogens. Disinfect the tools used before grafting. The scion is grafted as soon as it is picked. When picking, the leaves are immediately cut off, the labels are hung, and they are wrapped with wet towels and put in plastic bags. The bag mouth is left for ventilation and can be stored for 2 days. The commonly used rootstocks in Guangxi: Ziyang orange and Citrus aurantium are used as rootstocks. In our previous research, it was found that the comprehensive evaluation of cold resistance and salt-alkali resistance of Citrus aurantium is better than that of Ziyang orange. The method for obtaining the rootstock is to cultivate the rootstock by sowing Citrus aurantium or Ziyang orange seeds. After the rootstocks were cultivated, the scions were grafted onto Fructus Aurantii Immaturus and Citrus citrus fruit using a conventional grafting method. The grafted scion and rootstock combinations are shown in Table 2. After one year of cultivation in the nursery bed, 10 grafted seedlings of each combination with good growth were planted in an epidemic area seriously infected with Huanglongbing. Six months later, the grafted seedlings were tested according to the methods of Example 1 and PCR, respectively, wherein the nitrogen-green ratio and the sugar-green ratio were averaged, and as long as one of the 10 parallel grafted seedlings in each group was detected to be positive by PCR, the group of grafted seedlings was evaluated as positive. The specific test results are shown in Table 3.

[0036] Table 2 Combinations of grafted seedlings with different scion rootstocks

[0037] Group Scion Rootstock Group Scion Rootstock Grafted seedlings 1 GJ-1-1 Citrus aurantium Grafted seedlings 11 GJ-1-1 Ziyang Orange Grafted seedlings 2 GJ-1-3 Citrus aurantium Grafted seedlings 12 GJ-1-3 Ziyang Orange Grafted seedlings 3 GJ-3-1 Citrus aurantium Grafted seedlings 13 GJ-3-1 Ziyang Orange Grafted seedlings 4 GJ-3-4 Citrus aurantium Grafted seedlings 14 GJ-3-4 Ziyang Orange Grafted seedlings 5 GJ-3-5 Citrus aurantium Grafted seedlings 15 GJ-3-5 Ziyang Orange Grafted seedlings 6 GJ-4-1 Citrus aurantium Grafted seedlings 16 GJ-4-1 Ziyang Orange Grafted seedlings 7 GJ-4-3 Citrus aurantium Grafted seedlings 17 GJ-4-3 Ziyang Orange Grafted seedlings 8 GJ-7-2 Citrus aurantium Grafted seedlings 18 GJ-7-2 Ziyang Orange Grafted seedlings 9 GJ-7-3 Citrus aurantium Grafted seedlings 19 GJ-7-3 Ziyang Orange Grafted seedlings 10 GJ-7-4 Citrus aurantium Grafted seedlings 20 GJ-7-4 Ziyang Orange

[0038] Results of the Huanglongbing resistance test 6 months after transplanting:

[0039] Table 3 The infection of Huanglongbing in different grafted seedling disease areas after transplantation

[0040]

[0041]

[0042] As shown in Table 3, the same scion grafted onto different rootstocks has different disease resistance. There is 1 group of positive grafted seedlings on the Citrus aurantium rootstock, and 6 groups of positive grafted seedlings on the Ziyang orange. This shows that the Citrus aurantium rootstock with better cold resistance and salt and alkali resistance has stronger survival ability in the Huanglongbing area, and can fully transport nutrients to the scion branches to maintain the high resistance of the branches to Huanglongbing. However, since the salt and alkali resistance and cold resistance of Ziyang orange are not as good as those of Citrus aurantium, it is easily affected by environmental conditions in the Huanglongbing area after grafting, which affects the performance of the rootstock. After the rootstock cannot transport nutrients upward well, the resistance of the grafted seedlings decreases, and it is more susceptible to the Huanglongbing pathogen.

[0043] In addition, through observation of these positive grafted seedlings, it was found that their scions were reflected in Table 1 as sub-healthy branches that did not contain Huanglongbing pathogens with nitrogen-green ratio (N / SP)>0.150 or sugar-green ratio (T / SP)<0.10, indicating that sub-healthy branches in the Huanglongbing area are more likely to be infected with Huanglongbing than healthy branches that do not contain Huanglongbing pathogens with nitrogen-green ratio (N / SP)>0.150 and sugar-green ratio (T / SP)<0.10 if the nutrient transport of the rootstock is poor.

[0044] From the experimental results in Table 3, it can be seen that when the nitrogen-green ratio (N / SP) ≤ 0.150 and the sugar-green ratio (T / SP) ≥ 0.10, the plant is infected with Huanglongbing; when the nitrogen-green ratio (N / SP) > 0.150 and / or the sugar-green ratio (T / SP) < 0.10, the method for determining that the plant is not infected with Huanglongbing is still valid.

[0045] In summary, the present invention is combined with previous research, and a model for determining whether citrus is infected with Huanglongbing can be constructed only through test data from a handheld chlorophyll meter and a handheld plummeter. The model is: when the nitrogen-green ratio is ≤0.150 and the sugar-green ratio is ≥0.10, the plant is infected with Huanglongbing; when the nitrogen-green ratio is >0.150 and / or the sugar-green ratio is <0.10, the plant is not infected with Huanglongbing; after PCR verification, the conclusion of the model is consistent with the PCR result. The construction of this model provides a reliable and convenient method for the rapid initial judgment of Huanglongbing. In addition, through grafting experiments, it is proved that citrus varieties and / or strains with a nitrogen-green ratio >0.150 and a sugar-green ratio <0.10 can be used as varieties and / or strains with high resistance to Huanglongbing.

[0046] The above examples only express several embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for quickly determining the susceptibility of citrus Huanglongbing based on a citrus Huanglongbing evaluation model, characterized in that: The method comprises the following steps: randomly taking leaves from citrus plants, using a handheld chlorophyll meter and a handheld pliometer to measure the SPAD value, nitrogen content and sucrose content of the leaves, and then calculating the nitrogen-green ratio and the sugar-green ratio; when the nitrogen-green ratio is ≤0.150 and the sugar-green ratio is ≥0.10, the plants are infected with Huanglongbing; when the nitrogen-green ratio is >0.150 and / or the sugar-green ratio is <0.10, the plants are not infected with Huanglongbing; The nitrogen-green ratio is calculated as follows: leaf nitrogen content / SPAD value, and the sugar-green ratio is calculated as follows: leaf brix / SPAD value.

2. The method according to claim 1, characterized in that The leaf sampling method is as follows: 5 different branches are taken from each tree, 8 leaves are taken from each branch, 3 leaves are taken from the top leaves of the new shoots, and 5 leaves are taken from each of the 3rd layers from the top.

3. A method for breeding varieties and / or strains with high resistance to Huanglongbing based on a citrus Huanglongbing evaluation model, characterized in that: The method comprises: randomly taking leaves from citrus plants in a Huanglongbing diseased area, measuring the SPAD value, nitrogen content and sucrose content of the leaves using a handheld chlorophyll meter and a handheld pliometer, and then calculating the nitrogen-green ratio and the sugar-green ratio, and breeding citrus varieties and / or strains with a nitrogen-green ratio greater than 0.150 and a sugar-green ratio less than 0.10; The nitrogen-green ratio is calculated as follows: leaf nitrogen content / SPAD value, and the sugar-green ratio is calculated as follows: leaf brix / SPAD value.

4. A method for breeding varieties and / or strains with high resistance to Huanglongbing based on a citrus Huanglongbing evaluation model, characterized in that: The method comprises the following steps: randomly taking leaves from citrus plants, measuring the SPAD value, nitrogen content and sucrose content of the leaves using a handheld chlorophyll meter and a handheld pliometer, then calculating the nitrogen-green ratio and the sugar-green ratio, selecting citrus plant branches with a nitrogen-green ratio greater than 0.150 and a sugar-green ratio less than 0.10 as scions, and performing grafting or cross breeding on citrus; the grafted rootstock is Citrus aurantium; The nitrogen-green ratio is calculated as follows: leaf nitrogen content / SPAD value, and the sugar-green ratio is calculated as follows: leaf brix / SPAD value.

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