WO2014201744A1 - 一种柑橘黄龙病快速诊断方法 - Google Patents

一种柑橘黄龙病快速诊断方法 Download PDF

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Publication number
WO2014201744A1
WO2014201744A1 PCT/CN2013/079470 CN2013079470W WO2014201744A1 WO 2014201744 A1 WO2014201744 A1 WO 2014201744A1 CN 2013079470 W CN2013079470 W CN 2013079470W WO 2014201744 A1 WO2014201744 A1 WO 2014201744A1
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Prior art keywords
leaves
blade
citrus
cut
main vein
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PCT/CN2013/079470
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English (en)
French (fr)
Inventor
毛润乾
张宇宏
郑基焕
潘志萍
全金成
黄明度
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广东省昆虫研究所
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Priority to US14/898,965 priority Critical patent/US9921213B2/en
Priority to AU2013393172A priority patent/AU2013393172B2/en
Publication of WO2014201744A1 publication Critical patent/WO2014201744A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects

Definitions

  • the invention belongs to the field of citrus disease prevention and control, and particularly relates to a rapid diagnosis method for citrus Huanglong disease. Background technique:
  • Citrus pests and diseases are a major obstacle to the development of the citrus industry, the most serious of which is the citrus Huanglong disease known as citrus "cancer". With the rapid development of citrus production, the occurrence and damage of citrus Huanglongbing has become more and more serious.
  • the disease is a highly contagious disease with a long epidemic, a wide range of morbidity and a high incidence. It results in short life span, low yield and high production cost in most parts of China. The annual economic loss is as high as billions. The US dollar has seriously plagued and restricted the development of the citrus industry in China and the world.
  • detection and diagnosis techniques are mainly used for field symptom diagnosis and indication plant identification, electron microscopy detection, immunological detection and diagnosis, nucleic acid molecule detection and diagnosis, and diagnostic methods based on starch iodine reaction (Zhang Liping, 2009), in which PCR technology is the current mainstream technology ( Deng Xiaoling et al., 1999; Wang Zhongkang et al., 2004; Meng Xiangchun et al., 2007).
  • the diagnostic method based on starch iodine reaction originated from Schneider (1968). It was found that the starch content of the leaves infected by Huanglongbing was very high.
  • the present inventors have found through research that the current starch coloring technology is not widely used for the following reasons: (1) Since the morning sampling, the effect of normal leaf starch is not really eliminated. Normal leaves undergo photosynthesis to accumulate starch, sometimes due to environmental factors, may not be completely transferred at night, resulting in starch residues in normal leaves in the morning; (2) Because the above techniques do not remove chlorophyll, there is interference during color development, which may also affect the accuracy of detection and diagnosis. Rate; (3) The above diagnostic technique needs to be mixed with water for grinding, and the starch is insoluble in water, mixed with water for color reaction, and the accuracy is easily affected. Summary of the invention:
  • the method for rapid diagnosis of citrus Huanglongbing of the present invention comprises the following steps: a. Dark treatment: Covering the leaves of the citrus to be detected with a black bag, sealing, and darkening for 12-24 hours to obtain darkly treated leaves Or collect the branches of the citrus to be tested, bring them back into the room, insert them in water, cover the leaves with black bags or place them in a dark room, darkly treat them for 12-24 hours, collect the leaves to obtain darkly treated leaves; or Before exiting, collect the leaves of the citrus to be tested overnight as the dark treated leaves;
  • Freezing treatment of the leaves Shearing the leaves after dark treatment, at least cutting one side of the main vein of the blade, cutting from the side of the blade along the lateral vein of the blade to the main vein of the blade until the main vein, but not cutting the main Pulse, thus cutting the leaves into thin strips with a number of thin strips, then placed at -4 ⁇ - 15 °C frozen 12 ⁇ 24 small When the frozen leaves are obtained; or the darkly treated leaves are frozen at -4 to - 15 °C for 12 to 24 hours, and then the leaves are sheared, at least one side of the main vein of the blade is cut, from The lateral side of the blade is cut along the lateral vein of the blade to the main vein of the blade until the main vein, but the main vein is not cut, so that the blade is cut into thin strips with several thin strips to obtain the frozen leaf;
  • Color detection Add starch chromogenic solution on the strips of the decolorized leaves, and then observe the color change of the leaves. If the leaves turn blue, the leaves of the citrus to be detected are infected with Huanglong disease, if the leaves are not discolored. , then normal leaves.
  • the chlorophyll decolorizing liquid refers to a solution capable of removing chlorophyll, and is preferably a mixture of one or more of an organic solvent such as acetone, ethanol, benzene, toluene or xylene.
  • the starch coloring liquid refers to a solution capable of developing colored starch, and is preferably iodine, potassium iodide solution or polyvinylpyrrolidone iodine.
  • the strip is preferably a strip of 1 mm width.
  • the starch in the normal leaves is transferred by dark treatment, and no starch is accumulated in the leaves.
  • the screen of the Huanglong disease is blocked by the screen, the starch cannot be transferred and deposited in the leaves. Therefore, the dark treatment can truly eliminate the influence of the normal leaf starch and improve the accuracy.
  • the main purpose of the leaf freezing treatment is to remove chlorophyll, because the chlorophyll is not completely removed in the prior art, and there is interference in color development, which also affects the accuracy of detection and diagnosis.
  • the invention freezes, the fusion at temperature accelerates the rupture of the cell membrane, thereby shortening the leaching time of chlorophyll, and by cutting the leaves into thin strips, the chlorophyll can be accelerated, but not completely cut, and the shape of the leaves is maintained. Conducive to the subsequent steps, through the above steps to accelerate the dissolution of chlorophyll, and help the chlorophyll dissolve completely to remove chlorophyll.
  • the decolorization of the leaves is to dissolve and extract the chlorophyll in the leaves by using the chlorophyll decolorizing solution until the leaves become white, thereby completely removing the chlorophyll, avoiding the interference due to the presence of chlorophyll, and affecting the accuracy of detection and diagnosis.
  • the color detection is based on the principle that the leaves of Huanglong disease are rich in starch, the starch is iodine-blue, and the normal leaves do not contain starch, and the color is unchanged.
  • the above-mentioned decolorized leaves are detected and diagnosed by using starch coloring solution, thereby determining Check if the leaves are yellow dragon disease leaves.
  • Table 1 The difference between the rapid diagnosis method of the citrus Huanglong disease of the present invention and the prior art is shown in Table 1:
  • the shape of the blade is ground and then slurried.
  • the leaves are cut into thin strips, the main veins are continuous, and the overall shape of the leaves is unchanged.
  • Color development, color development on the reaction film, color development directly on the blade is unchanged.
  • the invention effectively removes the residual starch in the leaves, removes the influence of residual starch, and then removes the chlorophyll effectively, removes the influence of chlorophyll, directly develops color on the leaves, does not need to be mixed with water, thereby avoiding the starch being insoluble.
  • accuracy is easily affected, which greatly improves the diagnostic accuracy of citrus Huanglongbing, and achieves a rapid diagnosis of citrus Huanglongbing, which provides effective prevention and treatment of Huanglongbing. Detection means, is conducive to the prevention and treatment of Huanglong disease.
  • FIG. 1 is a schematic view of three cases in which the blade is cut into thin strips. A indicates that only one side of the main vein is cut, B indicates that both sides of the main vein are sheared, and C indicates that one side of the main vein is removed. Cutting the remaining one side blade;
  • FIG. 2 is a diagnosis result diagram of Embodiment 1;
  • Figure 3 is a diagram showing the diagnosis results of Example 2.
  • Figure 4 is a diagram showing the diagnosis results of Example 3.
  • Figure 5 is a diagram showing the diagnosis results of Example 4.
  • Figure 6 is a diagram showing the diagnosis results of Example 5.
  • Figure 7 is a diagram showing the diagnosis results of Example 6;
  • Fig. 8 is a graph showing the results of diagnosis of Example 7. detailed description:
  • Example 1 Detection and diagnosis of granulated huanglongbing disease.
  • the pulse direction is cut to the main vein of the blade until the main vein, but the main vein is not cut, so that the blade is cut into thin strips with a number of strips of width lmm, as shown in Fig. 1A), placed in the frozen layer of the refrigerator, and the freezing temperature is frozen. It is -10 ° C.
  • the frozen leaves were taken at 8:00 am on the 14th, placed in a test tube, and 25 mL of chlorophyll decoloring solution (acetone) was added, and shaken 2-3 times in the middle. At 3 o'clock in the afternoon, the thin blades turned white, and the test tube decolorization liquid was poured off to obtain the decolorized leaves.
  • the starch coloring liquid (iodine) was directly dropped on the strips of the decolored leaves, and the change in the color of the leaves was observed for diagnosis.
  • the results are shown in Fig. 2.
  • a in Fig. 2 is the blade to be detected of the present embodiment, and B is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf. It can be seen from FIG. 2 that after the coloration of the blade (A) to be detected in this embodiment is performed, the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, and the normal leaf (B) is not discolored. , thus identified as normal leaves that do not suffer from Huanglongbing. The detection result is consistent with the detection result using the conventional nucleic acid molecule detection.
  • Example 2 Detection and diagnosis of horse water orange dragon disease
  • the thin strips of the leaves turn white, pour off the decolorizing liquid, take out the leaves, obtain the decolorized leaves, and add the starch coloring solution on the strips of the decolorized leaves ( Potassium iodide solution) Diagnosis.
  • a in Fig. 3 is the blade to be detected of the present embodiment, and B is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf.
  • the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, and the normal leaf (B) is not discolored. , thus identified as normal leaves that do not suffer from Huanglongbing.
  • the detection result is consistent with the detection result using the conventional nucleic acid molecule detection.
  • Example 3 Detection and diagnosis of Luogang orange yellow dragon disease.
  • the side edge is cut along the lateral vein of the blade to the main vein of the blade until the main vein, but the main vein is not cut, so that the blade is cut into strips with a number of strips of width lmm, as shown in Fig. 1A), placed in the frozen layer of the refrigerator. Freeze, freezing temperature is -4 °C.
  • freezing temperature is -4 °C.
  • the frozen leaf pieces were taken out, placed in a test tube, and 25 mL of chlorophyll decolorizing solution (acetone) was added, and the mixture was shaken 2-3 times.
  • the thin strips turned white, and the test tube decolorization liquid was poured off to obtain the decolorized leaves.
  • the starch coloring liquid (iodine) was directly dropped on the strips of the decolored leaves, and the change in the color of the leaves was observed for diagnosis.
  • FIG. 4 B in Fig. 4 is the blade to be detected of the present embodiment, and A is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf. It can be seen from FIG. 4 that after the coloration of the blade (B) to be inspected in this embodiment, the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, while the normal leaf (A) does not change color. , thus identified as normal leaves that do not suffer from Huanglongbing. This test result is consistent with the detection of diagnostic results using conventional nucleic acid molecules.
  • Example 4 Detection and diagnosis of navel orange yellow dragon disease
  • FIG. 5 The result is shown in Fig. 5.
  • B and C in Fig. 5 are the blades to be detected of the embodiment, and A is already It is proved by the prior art that it is not a normal leaf of the yellow dragon disease leaf.
  • the thin strips of the leaves are blue, thereby identifying that the leaves to be detected are Huanglong diseased leaves, while the normal leaves (A) are It does not change color, and thus is identified as a normal leaf that does not suffer from Huanglongbing.
  • the detection result is consistent with the detection result using the conventional nucleic acid molecule detection.
  • Example 5 Detection and diagnosis of Hongjiang orange yellow dragon disease
  • the branches of Hongjiang Orange to be tested were collected at 1 pm on May 23, 2013, brought back indoors, the branches were inserted in the water, and placed in the dark room at 8 am on the 24th. After dark treatment, the darkened leaves were obtained, and then placed in a freezer layer to freeze at a temperature of -4 V. After 24 hours, the leaves were cut into strips of about 1 mm width (the blade side was from the blade side). The edge of the blade is cut toward the main vein of the blade, until the main vein, but the main vein is not cut, so that the blade is cut into strips with a number of strips of width lmm, as shown in Fig. 1A), and the frozen leaves are obtained.
  • FIG. 6 A in Fig. 6 is the blade to be detected of the present embodiment, and B is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf. It can be seen from FIG. 6 that after the coloration of the blade (A) to be detected in this embodiment is performed, the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, and the normal leaf (B) does not change color. , thus identified as normal leaves that do not suffer from Huanglongbing. This test result is consistent with the detection of diagnostic results using conventional nucleic acid molecules.
  • Example 6 Detection and diagnosis of summer orange yellow dragon disease
  • FIG. 7 A in Fig. 7 is the blade to be tested of the present embodiment, and B is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf. It can be seen from FIG. 7 that after the coloration of the blade (A) to be detected in this embodiment is performed, the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, and the normal leaf (B) is not discolored. , thus identified as normal leaves that do not suffer from Huanglongbing. This test result is consistent with the detection of diagnostic results using conventional nucleic acid molecules.
  • Example 7 Detection and diagnosis of Huangdan Huanglong disease.
  • the branches of the emperor to be tested were collected in the Deqing Citrus Garden, brought back into the room, the branches were inserted in the water, and the leaves were covered with black bags for dark treatment, and dark treatment was carried out for 24 hours.
  • the leaves after dark treatment (the leaves to be detected, diagnosed by nucleic acid molecules, which are Huanglong disease leaves), the darkly treated leaves were placed in the freezer layer for freezing for 14 hours at 6 pm on May 30, and the freezing temperature was -10 °C, at 8 am on May 31, the leaves are cut into strips about 1 mm wide (the blade side is cut from the side of the blade along the lateral vein of the blade to the main vein of the blade, until the main vein, but the main vein is not cut, so that the blade Cut into a thin strip with a number of strips of width lmm, as shown in Fig.
  • FIG. 8 B in Fig. 8 is the blade to be detected of the present embodiment, and A is a normal leaf which has been proved by the prior art to be not a yellow dragon disease leaf. It can be seen from FIG. 8 that after the coloration of the blade (B) to be detected in this embodiment is performed, the thin strip-shaped blade is blue, thereby identifying that the leaf to be detected is a yellow dragon disease leaf, and the normal leaf (A) is not discolored. , thus identified as normal leaves that do not suffer from Huanglongbing. The test results and use often

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Abstract

一种柑橘黄龙病快速诊断方法，包括暗处理、叶片的冷冻、脱色及显色检测步骤。该方法通过去除叶片中叶绿素以及残留淀粉，实现直接在叶片上显色，无需与水混合研磨，有利于柑橘黄龙病的快速准确诊断。

Description

一种柑橘黄龙病快速诊断方法
技术领域：
本发明属于柑橘病害防治领域， 具体涉及一种柑橘黄龙病快速诊断方法。 背景技术：
我国是世界上的柑橘生产大国； 柑橘产业是我国农业支柱产业， 在农业经济 中占有极其重要的地位，在促进农民增收、增加社会就业和社会主义新农村建设 中发挥极其重要的作用。柑橘病虫害是柑橘产业发展的重大障碍， 其中最严重的 是被称为柑橘 "癌症 "的柑橘黄龙病。 随着柑橘生产的快速发展， 柑橘黄龙病的发 生和为害越来越严重。 该病是一种为害极大的传染性病害， 其流行持续时间长、 发病范围广、发病率高，造成我国大部分地区柑橘寿命短、产量低、生产成本高， 每年经济损失高达数十亿美元，严重困扰和制约我国乃至世界范围内柑橘产业的 发展。
研究结果已证明， 田间的黄龙病病菌主要是由柑橘木虱传播的。 因此， 防治 柑橘木虱， 减少田间柑橘木虱的种群数量， 成为控制黄龙病的核心问题。广东省 昆虫研究所在柑橘木虱和柑橘黄龙病方面进行了比较深入的研究，并申请发明专 利"一种柑橘园控制柑橘木虱和黄龙病的方法"， 申请号： 201310049098.6。
然而， 目前黄龙病防治面临的主要问题是难度大，技术要求高，实施较困难。 其问题的关键是由于检测诊断技术跟不上，种植户不认为自己的柑橘树染上黄龙 病， 所以也就不积极去采取措施。 因此， 快速准备易操作的检测诊断技术， 成为 黄龙病防治的急需的技术难点。
目前， 检测诊断技术主要田间症状诊断及指示植物鉴别、 电镜检测诊断、免 疫学检测诊断、核酸分子检测诊断、基于淀粉碘反应的诊断方法（张利平， 2009)， 其中 PCR技术是目前的主流技术 （邓晓玲等， 1999; 王中康等， 2004; 孟祥春 等， 2007)。 基于淀粉碘反应的诊断方法来源于 Schneider (1968) 发现黄龙病感 染的叶片中淀粉含量很高， 2002年 Masatoshi Onuki用组织学方法证实了感染 黄龙病的柑橘叶片积累大量的淀粉粒。 Le Thi Thu Hong和 Nguyen Thi Ngoc True (2003 )随后建立了碘反应诊断黄龙病技术， 技术要点为： （1 )采集黄龙病特征 的叶片， 采集时不要采集内膛叶、 卷叶、 嫩叶， 采集时间为早晨， 不要采集枝、 果和根。 （2)取 lg的叶片， 与 2 ml蒸馏水混合， 研碎。 （3 )滴加 2 L叶和水的 混合液在反应膜上 CNCM， Nitrocellulose Membrane), 5分钟后， 滴加 2 L碘液 在反应膜的样品上。 （4) 观察叶片颜色变化， 等待结果， 阴性反应颜色不变，阳 性则变蓝。 日本 TAKUSHI(2007)测定感染黄龙病的柑橘叶片中淀粉含量是 400-500mg/kg， 正常叶片为 85.6 mg/kg， 并建立快速诊断技术， 他们用砂纸擦刮 叶片表面 20下， 然后将砂纸放在塑料袋中， 向袋内加 25 L的碘液显色。 马来 西亚的 Lily Eng (2007) 用不同的砂纸重复了上述方法。 我国张利平等 （2009) 重复该技术， 磨碎采集的柑橘感病材料， 加等量的纯水， 然后吸取 4 L叶片和 水混合液于反应膜上， 等 5 分钟， 滴加等量的碘溶液， 观察颜色变化， 试验结 果与 PCR结果一致性可以达到 93.3%。
本发明人通过研究发现： 目前淀粉显色技术之所以没有广泛应用是因为以下 原因， （1 ) 由于早晨采样， 没有真正消除正常叶淀粉的影响。 正常叶进行光合作 用积累淀粉， 有时由于环境因素， 晚上可能不能完全转移， 导致早晨正常叶中有 淀粉残留； （2) 由于上述技术没有去掉叶绿素， 显色时存在干扰， 也可能影响检 测诊断准确率； （3 )上述诊断技术中需要与水混合进行研磨， 而淀粉是不溶于水 的， 与水混合进行显色反应， 准确性易受影响。 发明内容：
本发明的目的是提供一种简便快速， 准确性高的柑橘黄龙病快速诊断方 法。
本发明的柑橘黄龙病快速诊断方法， 其特征在于， 包括以下步骤： a、 暗处理： 用黑色袋子套住待检测的柑橘的叶片， 封口， 暗处理 12-24 小时， 得到暗处理后的叶片； 或者采集待检测的柑橘的枝条， 带回室内， 插在 水中， 再用黑色袋子套住叶片或放在暗室中， 暗处理 12-24小时， 收集叶片得 到暗处理后的叶片； 或者在日出之前， 去采集过夜的待检测的柑橘的叶片作为 暗处理后的叶片；
b、 叶片的冷冻处理： 对暗处理后的叶片进行剪切， 至少剪切叶片主脉的 一侧叶片， 从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断 主脉， 从而将叶片剪成具有若干细条的细条状， 再置于 -4〜- 15°C冷冻 12〜24小 时，得到冷冻后的叶片；或者将暗处理后的叶片置于 -4〜- 15 °C冷冻 12〜24小时， 然后再对叶片进行剪切， 至少剪切叶片主脉的一侧叶片， 从叶片侧边沿叶片侧 脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干 细条的细条状， 得到冷冻后的叶片；
c、 叶片的脱色： 将冷冻后的叶片放入叶绿素脱色液中， 直至叶片的细条 变为白色， 再取出， 得到脱色后的叶片；
d、 显色检测： 在脱色后的叶片的细条上滴加淀粉显色液， 然后观察叶片 的颜色变化， 如果叶片变蓝， 说明待检测的柑橘的叶片为黄龙病感染叶， 如果 叶片不变色， 则为正常叶。
所述的叶绿素脱色液是指能脱除叶绿素的溶液， 优选为丙酮、 乙醇、 苯、 甲苯或二甲苯等有机溶剂中的一种或几种的混合物。
所述的淀粉显色液是指能显色淀粉的溶液， 优选为碘酒、碘化钾溶液或聚 乙烯吡咯烷酮碘。
所述的细条优选为 1mm宽的细条。
本发明通过暗处理， 正常叶片中的淀粉被转移， 叶片中无淀粉积累。而黄龙 病叶因为筛管堵塞， 淀粉无法转移， 沉积在叶片中， 因此通过暗处理可以真正的 消除正常叶淀粉的影响， 提高准确率。
叶片的冷冻处理，其主要目的是去除叶绿素， 因为现有技术中没有完全去除 叶绿素， 显色时会存在干扰， 也影响检测诊断准确率。 本发明通过冷冻处理，温 度上的聚变加速细胞膜的破裂， 从而縮短叶绿素的溶解浸提时间， 再加上对叶片 剪切成细条状， 可以加速叶绿素的溶解， 但不完全剪断， 保持叶片形状， 有利于 后续步骤的进行，通过上述步骤加速叶绿素的溶解， 并有助于叶绿素溶解完全从 而去除叶绿素。
叶片的脱色是利用叶绿素脱色液溶解和萃取叶片中的叶绿素，直至叶片变白 色， 从而完全去除叶绿素， 避免由于叶绿素的存在形成干扰， 影响检测诊断准确 率。
显色检测是利用黄龙病叶富含淀粉， 淀粉遇碘变蓝， 而正常叶内不含淀粉， 颜色不变的原理，利用淀粉显色液对上述脱色后的叶片进行检测诊断， 从而确定 待检测叶片是否是黄龙病叶的。 本发明的柑橘黄龙病快速诊断方法与现有技术的不同之处如表 1所示:
表 1
技术内容 现有技术中的显色技术 本发明 研磨叶片 需要 不需要
暗处理 无 有
脱色 （去叶绿素） 无 有
叶片形状 研磨后， 呈浆状。 叶片剪成细条， 中间主脉不断， 叶片总体形态不变。 显色情况 在反应膜上显色 直接在叶片上显色
与 PCR检测一致性 93.3% 99.9%
本发明通过有效的去除叶片中的残留淀粉， 去除残留淀粉的影响， 再通过有 效的去除叶绿素， 去除叶绿素的影响， 直接在叶片上显色， 无需与水混合研磨， 从而避开由于淀粉是不溶于水的， 与水混合进行显色反应， 准确性易受影响，从 而大大的提高了柑橘黄龙病的诊断准确性， 实现了对柑桔黄龙病的快速诊断，为 黄龙病的防治提供了有效检测手段， 有利于黄龙病的防治。 附图说明：
图 1是剪切叶片成细条的三种情况示意图， A表示只剪切主脉的一侧叶片， B表 示主脉的两侧叶片都剪切， C表示去除主脉的一侧叶片后，剪切剩余的一侧叶片； 图 2是实施例 1的诊断结果图；
图 3是实施例 2的诊断结果图；
图 4是实施例 3的诊断结果图；
图 5是实施例 4的诊断结果图；
图 6是实施例 5的诊断结果图；
图 7是实施例 6的诊断结果图；
图 8是实施例 7的诊断结果图。 具体实施方式：
以下实施例是对本发明的进一步说明， 而不是对本发明的限制。
实施例 1 : 沙糖桔黄龙病的检测诊断。
在广州萝岗柑橘园中进行， 时间 2013年 4月 12日 10: 00暗处理 （用黑色 胶袋将待检测沙糖桔树叶片（待检测叶片， 经核酸分子检测诊断， 其为黄龙病感 染叶） 罩住， 用夹子夹紧封口）。 13日上午 9: 30采集叶片， 得到暗处理后的叶 片，放在黑色胶袋中带回室内，下午 4点在室内将叶片剪成约 1mm宽的细条（叶 片一侧从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干宽度为 lmm细条的细条状， 具体如图 1A所示）， 放入 冰箱冷冻层冷冻， 冷冻温度为 -10°C。 14日上午 8 : 00取出冷冻后的叶片， 放试 管中， 加入叶绿素脱色液 （丙酮） 25mL， 中间摇动 2-3次。 下午 3点细条叶片 变为白色， 将试管脱色液倒掉， 得到脱色后的叶片， 在脱色后的叶片的细条上直 接滴加淀粉显色液 （碘酒）， 观察叶片颜色的变化进行诊断。 结果如图 2所示， 图 2中的 A为本实施例的待检测叶片， 而 B是已经通过现有技术证明其不是黄 龙病叶的正常叶。 由图 2可以看出， 本实施例的待检测叶片 （A) 经显色后， 细 条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而正常叶（B )则不变色， 由此鉴定为不患黄龙病的正常叶。该检测结果与利用常规的核酸分子检测诊断检 测结果一致。 实施例 2: 马水桔黄龙病的检测诊断
在阳春果园中进行， 下午 3点暗处理（用黑色袋子套住待检测的马水桔的叶 片 （经核酸分子检测诊断， 其为黄龙病叶） 罩住， 用夹子夹紧封口）， 第二天早 上拿出， 得到暗处理后的叶片， 然后再放入冰箱冷冻层， -15 温度, 冷冻 12小 时后取出， 然后将叶片剪成约 lmm宽的细条 （叶片一侧从叶片侧边沿叶片侧脉 方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干宽度 为 lmm细条的细条状， 具体如图 1A所示）， 得到冷冻后的叶片， 将冷冻后的叶 片放入叶绿素脱色液（乙醇） 中， 脱色 6小时后， 叶片细条变为白色， 倒掉脱色 液，取出叶片，得到脱色后的叶片，再脱色后的叶片的细条上滴加淀粉显色液（碘 化钾溶液） 诊断。
结果如图 3所示， 图 3中的 A为本实施例的待检测叶片， 而 B是已经通过 现有技术证明其不是黄龙病叶的正常叶。 由图 3可以看出， 本实施例的待检测叶 片 （A) 经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而 正常叶 （B ) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果与利用常 规的核酸分子检测诊断检测结果一致。 实施例 3: 萝岗橙黄龙病的检测诊断。
在广州萝岗萝岗橙园中进行， 时间 2013年 4月 12日 10: 00暗处理 （用黑 色胶袋将待检测萝岗橙树叶片（待检测叶片， 经核酸分子检测诊断， 其为黄龙病 叶）罩住， 用夹子夹紧封口）。 13日上午 9: 30采集叶片， 得到暗处理后的叶片， 放在黑色胶袋中带回室内， 下午 4点在室内将暗处理后的叶片剪成约 lmm宽的 细条（叶片一侧从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪 断主脉， 从而将叶片剪成具有若干宽度为 lmm细条的细条状， 具体如图 1A所 示）， 放入冰箱冷冻层冷冻， 冷冻温度为 -4°C。 14日上午 8: 00取出冷冻后的叶 片， 放试管中， 加入叶绿素脱色液 （丙酮） 25mL， 中间摇动 2-3次。 下午 3点 细条叶片变为白色， 将试管脱色液倒掉， 得到脱色后的叶片， 在脱色后的叶片的 细条上直接滴加淀粉显色液 （碘酒）， 观察叶片颜色的变化进行诊断。
结果如图 4所示， 图 4中的 B为本实施例的待检测叶片， 而 A是已经通过 现有技术证明其不是黄龙病叶的正常叶。 由图 4可以看出， 本实施例的待检测叶 片 （B) 经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而 正常叶 （A) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果与利用常 规的核酸分子检测诊断检测结果一致。 实施例 4: 脐橙黄龙病的检测诊断
在韶关乐昌浩天果园中进行， 时间 2013年 5月 15-17日。 15日下午 5点暗 处理 （用黑色胶袋将待检测脐橙叶片罩住， 用夹子夹紧封口）。 16日上午 6: 00 采集经过暗处理后的叶片（待检测叶片， 经核酸分子检测诊断， 其为黄龙病叶）， 得到暗处理后的叶片， 带回室内 （采样点距室内距离约 100M), 放入冰箱冷冻 层冷冻， 温度为 -4V , 24小时后取出， 将叶片剪成约 lmm宽的细条 （叶片一侧 从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将 叶片剪成具有若干宽度为 lmm细条的细条状， 具体如图 1A所示）， 得到冷冻后 的叶片， 放试管中， 加入叶绿素脱色液 （甲苯） 25mL， 中间摇动 2-3次， 10小 时后， 细条叶片变为白色叶片， 将试管脱色液倒掉， 得到脱色后的叶片， 在脱色 后的叶片的细条上直接滴加淀粉显色液（碘酒）， 观察叶片颜色的变化进行诊断。
结果如图 5所示， 图 5中的 B和 C为本实施例的待检测叶片， 而 A是已经 通过现有技术证明其不是黄龙病叶的正常叶。 由图 5可以看出， 本实施例的待检 测叶片 （B和 C)经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙 病叶， 而正常叶 （A) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果 与利用常规的核酸分子检测诊断检测结果一致。 实施例 5: 红江橙黄龙病的检测诊断
在廉江青平果园进行，时间 2013年 5月 23日下午 1点采集待检测的红江橙 的枝条， 带回室内， 将枝条插在水中， 再放在暗室中， 24日上午 8点， 采集经 过暗处理的叶片， 得到暗处理后的叶片， 再将其放入冰箱冷冻层冷冻， 温度为 -4 V， 24小时后取出， 将叶片剪成约 lmm宽的细条（叶片一侧从叶片侧边沿叶片 侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干 宽度为 lmm细条的细条状，具体如图 1A所示），得到冷冻后的叶片，放试管中， 加入叶绿素脱色液（甲苯） 25mL， 中间摇动 2-3次， 6小时后， 细条叶片变为白 色， 倒掉叶绿素脱色液， 滴加淀粉显色液 （碘酒）， 观察叶片颜色的变化进行诊 断。
结果如图 6所示， 图 6中的 A为本实施例的待检测叶片， 而 B是已经通过 现有技术证明其不是黄龙病叶的正常叶。 由图 6可以看出， 本实施例的待检测叶 片 （A) 经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而 正常叶 （B) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果与利用常 规的核酸分子检测诊断检测结果一致。 实施例 6: 夏橙黄龙病的检测诊断
在北海夏橙园中采样， 时间 2013年 5月 25日下午暗处理（用黑色胶袋将待 检测夏橙叶片罩住， 用夹子夹紧封口）， 5月 26日上午 7点采集经过暗处理后的 叶片 （待检测叶片， 经核酸分子检测诊断， 其为黄龙病叶）， 放入冰箱冷冻层冷 冻， 温度为 -12°C， 5月 27日上午 6点取出， 将叶片剪成约 lmm宽的细条 （叶 片一侧从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干宽度为 lmm细条的细条状， 具体如图 1A所示）， 得到 冷冻后的叶片， 放试管中， 加入叶绿素脱色液 （甲苯） 25mL， 中间摇动 2-3次， 中午 12: 00细条叶片变为白色叶片， 在脱色后的叶片的细条上直接滴加淀粉显 色液 （碘酒）， 观察叶片颜色的变化进行诊断。
结果如图 7所示， 图 7中的 A为本实施例的待检测叶片， 而 B是已经通过 现有技术证明其不是黄龙病叶的正常叶。 由图 7可以看出， 本实施例的待检测叶 片 （A) 经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而 正常叶 （B) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果与利用常 规的核酸分子检测诊断检测结果一致。 实施例 7: 皇帝柑黄龙病的检测诊断。
时间 2013年 5月 29日，在德庆柑橘园中采集待检测的皇帝柑的枝条， 带回 室内， 将枝条插在水中， 再用黑色袋子套住叶片进行暗处理， 暗处理 24小时， 得到暗处理后的叶片 （待检测叶片， 经核酸分子检测诊断， 其为黄龙病叶）， 5 月 30日下午 6点将暗处理后的叶片放入冰箱冷冻层冷冻 14小时，冷冻温度为 -10 °C， 5月 31 日上午 8点将叶片剪成约 1mm宽的细条（叶片一侧从叶片侧边沿叶 片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若 干宽度为 lmm细条的细条状， 具体如图 1A所示）， 得到冷冻后的叶片， 放试管 中， 加入叶绿素脱色液 （甲苯） 25mL， 中间摇动 2-3次。 下午 3时细条叶片变 为白色叶片，得到脱色后的叶片， 在脱色后的叶片的细条上直接滴加淀粉显色液 (碘酒）， 观察叶片颜色的变化进行诊断。
结果如图 8所示， 图 8中的 B为本实施例的待检测叶片， 而 A是已经通过 现有技术证明其不是黄龙病叶的正常叶。 由图 8可以看出， 本实施例的待检测叶 片 （B) 经显色后， 细条状叶片呈蓝色， 由此鉴定该待检测叶片为黄龙病叶， 而 正常叶 （A) 则不变色， 由此鉴定为不患黄龙病的正常叶。 该检测结果与利用常

Claims

权利要求书
1、 一种柑橘黄龙病快速诊断方法， 其特征在于， 包括以下步骤： a、 暗处理： 用黑色袋子套住待检测的柑橘的叶片， 封口， 暗处理 12-24 小时， 得到暗处理后的叶片； 或者采集待检测的柑橘的枝条， 带回室内， 插在 水中， 再用黑色袋子套住叶片或放在暗室中， 暗处理 12-24小时， 收集叶片得 到暗处理后的叶片； 或者在日出之前， 去采集过夜的待检测的柑橘的叶片作为 暗处理后的叶片；
b、 叶片的冷冻处理： 对暗处理后的叶片进行剪切， 至少剪切叶片主脉的 一侧叶片， 从叶片侧边沿叶片侧脉方向向叶片主脉剪， 直至主脉， 但不要剪断 主脉， 从而将叶片剪成具有若干细条的细条状， 再置于 -4一 15°C冷冻 12~24小 时，得到冷冻后的叶片；或者将暗处理后的叶片置于 -4一 15°C冷冻 12~24小时， 然后再对叶片进行剪切， 至少剪切叶片主脉的一侧叶片， 从叶片侧边沿叶片侧 脉方向向叶片主脉剪， 直至主脉， 但不要剪断主脉， 从而将叶片剪成具有若干 细条的细条状， 得到冷冻后的叶片；
c、 叶片的脱色： 将冷冻后的叶片放入叶绿素脱色液中， 直至叶片的细条 变为白色， 再取出， 得到脱色后的叶片；
d、 显色检测： 在脱色后的叶片的细条上滴加淀粉显色液， 然后观察叶片的颜色 变化， 如果叶片变蓝， 说明待检测的柑橘的叶片为黄龙病感染叶， 如果叶片不变 色， 则为正常叶。
2、 根据权利要求 1所述的柑橘黄龙病快速诊断方法， 其特征在于， 所述的 叶绿素脱色液为丙酮、 乙醇、 苯、 甲苯或二甲苯中的一种或几种的混合物。
3、 根据权利要求 1所述的柑橘黄龙病快速诊断方法， 其特征在于， 所述的 淀粉显色液为碘酒、 碘化钾溶液或聚乙烯吡咯烷酮碘。
4、 根据权利要求 1所述的柑橘黄龙病快速诊断方法， 其特征在于， 所述的 细条为 1mm宽的细条。
PCT/CN2013/079470 2013-06-19 2013-07-16 一种柑橘黄龙病快速诊断方法 WO2014201744A1 (zh)

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