Rapid diagnosis method of citrus huanglongbing

Abstract

The present invention discloses a rapid diagnosis method of citrus huanglongbing. In the present invention, the accuracy of diagnosis of citrus huanglongbing is significantly improved in the following manners: eliminating the interference of residual starch by effectively removing the residual starch contained in the leaf; eliminating the interference of chlorophyll by effectively removing chlorophyll; and directly developing the color on the leaf rather than mixing and grinding the leaf in water, so as to avoid the problem that the accuracy of color development reaction conducted by mixing the leaf with water is liable to be interfered since the starch is insoluble in water. Therefore a rapid diagnosis of the citrus huanglongbing with much higher accuracy is achieved, which provides effective detection means for the control of the citrus huanglongbing and is beneficial for the control of the citrus huanglongbing.

Claims

1. A rapid diagnosis method of citrus huanglongbing, wherein the method comprises the following steps: a. conducting a dark treatment comprising; (a1) bagging a citrus leaf with a black bag, and sealing the black bag for 12-24 hours to obtain a dark-treated citrus leaf; (a2) collecting a citrus branch, bringing the citrus branch indoors and inserting the citrus branch into water, then bagging a citrus leaf of the citrus branch with a black bag, or placing the citrus branch in a dark room, for 12-24 hours, to obtain a dark-treated citrus leaf; or (a3) before sunrise, collecting an overnight citrus leaf as the dark-treated citrus leaf; b. a freezing treatment of the dark-treated citrus leaf comprising; (b1) clipping the dark-treated citrus leaf, wherein the dark-treated citrus leaf is clipped at least on one side of a main vein from a side edge of the dark-treated citrus leaf along a lateral vein direction towards the main vein and until the main vein is reached, without cutting off the main vein, so as to clip the dark-treated citrus leaf into several thin strips, and then freezing the thin strips under 4 to 15 C. for 12-24 hours to obtain a frozen citrus leaf; or (b2) freezing the dark-treated citrus leaf under 4 to 15 C. for 12-24 hours, then clipping the citrus leaf, wherein the citrus leaf is clipped at least on one side of a main vein from a side edge of the citrus leaf along a citrus leaf lateral vein direction towards the main vein and until the main vein is reached, without cutting off the main vein, so as to clip the citrus leaf into several thin strips, and thereby obtaining the frozen citrus leaf; c. a decoloration step comprising putting the frozen citrus leaf into a chlorophyll decoloring solution until the thin strips of the citrus leaf turn white, and then removing the citrus leaf from the chlorophyll decloring solution to obtain decolored thin strips of the citrus leaf; d. a detection step through color development comprising adding a starch color-development solution dropwise onto the decolored thin strips of the citrus leaf, and then observing the color change of the citrus leaf, wherein the citrus leaf turning blue indicates that the citrus leaf is infected with the citrus huanglongbing, and if the color of the citrus leaf is not changed, the citrus leaf is a normal citrus leaf.

2. The rapid diagnosis method of citrus huanglongbing of claim 1, wherein the chlorophyll decoloring solution is acetone, ethyl alcohol, benzene, methyl benzene or dimethyl benzene, or a mixture thereof.

3. The rapid diagnosis method of citrus huanglongbing of claim 1, wherein the starch color-development solution is an iodine tincture, potassium iodide solution, or polyvinylpyrrolidone-iodine.

4. The rapid diagnosis method of citrus huanglongbing of claim 1, wherein the thin strips are thin strips with a width of 1 mm.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram of three cases in which a leaf is clipped into thin strips, wherein A represents the case that the leaf is clipped only on one side of the main vein, B represents the case that the leaf is clipped on both sides of the main vein leaf; and C represents the case that one side of the leaf is removed, and the other side is clipped;

(2) FIG. 2 is a diagram showing the diagnosis result of embodiment 1;

(3) FIG. 3 is a diagram showing the diagnosis result of embodiment 2;

(4) FIG. 4 is a diagram showing the diagnosis result of embodiment 3;

(5) FIG. 5 is a diagram showing the diagnosis result of embodiment 4;

(6) FIG. 6 is a diagram showing the diagnosis result of embodiment 5;

(7) FIG. 7 is a diagram showing the diagnosis result of embodiment 6; and

(8) FIG. 8 is a diagram showing the diagnosis result of embodiment 7.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) The following embodiments are provided for further illustration of the present invention, and not for the purpose of limiting the invention.

(10) Embodiment 1: Detection and Diagnosis of Citrus Huanglongbing in Sugar Orange

(11) The embodiment is conducted in Luogang Citrus Orchard in Guangzhou. A dark treatment was performed at 10:00 on Apr. 12, 2013 [leaves of the sugar orange tree to be detected (the leaves to be detected were leaves infected with the citrus huanglongbing, as diagnosed through a detection of nucleic acid molecules) were bagged with black plastic bags, and then the bags were sealed with clamps]. At 9:30 a.m. on April 13.sup.th, the leaves were collected as dark-treated leaves, put into the black plastic bag and brought indoors. At 4:00 p.m., the leaves were clipped into thin strips with a width of 1 mm indoors (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), and then the leaves were frozen in the freezer of a refrigerator under a freezing temperature of 10 C. At 8:00 a.m. on April 14.sup.th, the frozen leaves were taken out and transferred into test tubes, and then a chlorophyll decoloring solution (acetone) of 25 ml was added into each test tube, wherein during the adding process, the tube was shaken for 2-3 times. At 3:00 p.m. on April 14.sup.th, the thin strips of leaves turned white, then the decoloring solution was poured out from the tube to obtain decolored leaves; and then a starch color-development solution (iodine tincture) was directly added onto the thin strips of the decolored leaves dropwise so as to perform a diagnosis by observing the color change of the leaves. The result is as shown in FIG. 2, wherein in FIG. 2, A is the leaf to be detected in this embodiment, and B is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 2 that after the leaf (A) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue, such that the leaf (A) to be detected is determined to be a leaf infected with citrus huanglongbing; and the color of the normal leaf (B) is not changed, such that the leaf (B) is determined to be a normal leaf not infected with citrus huanglongbing. This detection result is consistent with the detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(12) Embodiment 2: Detection and Diagnosis of Citrus Huanglongbing in Mashui Orange

(13) This embodiment is conducted in Yangchun Orchard. A dark treatment was performed at 3:00 p.m. [leaves of the Mashui orange to be detected (the leaves to be detected were leaves infected with citrus huanglongbing, as diagnosed through the detection of nucleic acid molecules) were bagged with black bags, and then the bags were sealed with clamps]. In the next morning, the leaves were taken out as dark-treated leaves, and then were frozen in the freezer of a refrigerator under a freezing temperature of 15 C. for 12 hours. Subsequently the leaves were clipped into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), so as to obtain frozen leaves. The frozen leaves were decolored in a chlorophyll decoloring solution (ethyl alcohol) for 6 hours and thus the thin strips of the leaf turned white. Decolored leaves were obtained by pouring out the decoloring solution and taking the leaves out. Thereafter a starch color-development solution (potassium iodide solution) was added onto the thin strips of the decolored leaves dropwise so as to perform a diagnosis.

(14) The result is as shown in FIG. 3, wherein in FIG. 3, A is the leaf to be detected in this embodiment, and B is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 3 that after the leaf (A) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue such that the leaf (A) to be detected is determined to be a leaf infected with the citrus huanglongbing; and the color of the normal leaf (B) is not changed such that the leaf (B) is determined to be a normal leaf (B) not infected with citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(15) Embodiment 3: Detection and Diagnosis of Citrus Huanglongbing in Luogang Citrus

(16) The embodiment is conducted in the Luogang Citrus Orchard in Luogang District, Guangzhou. A dark treatment was performed at 10:00 on Apr. 12, 2013 [leaves of the Luogang citrus tree to be detected (the leaves to be detected were leaves infected with the citrus huanglongbing, as diagnosed through detection of nucleic acid molecules) were bagged with black plastic bags, and then the bags were sealed with clamps]. At 9:30 a.m. on April 13.sup.th, the leaves were collected as dark-treated leaves, put in the black plastic bag and brought indoors. At 4:00 p.m. on April 13.sup.th, the dark-treated leaves were clipped indoors into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), and then were frozen in the freezer of a refrigerator under a freezing temperature of 4 C. At 8:00 a.m. on April 14.sup.th, the frozen leaves were taken out and transferred into test tubes, and then a chlorophyll decoloring solution (acetone) of 25 ml was added into each test tube, wherein during the addition, the tube was shaken for 2-3 times. At 3:00 p.m. on April 14.sup.th, the thin strips of leaves turned white, then the decoloring solution was poured out from the tube to obtain decolored leaves; and then a starch color-development solution (iodine tincture) was directly added onto the thin strips of the decolored leaves dropwise so as to perform a diagnosis by observing the color change of the leaves.

(17) The result is as shown in FIG. 4, wherein in FIG. 4, B is the leaf to be detected in this embodiment, and A is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 4 that after the leaf (B) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue, such that the leaf (B) to be detected is determined to be a leaf infected with the citrus huanglongbing; and the color of the normal leaf (A) is not changed, such that the leaf (A) is determined to be a normal leaf (A) not infected with the citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(18) Embodiment 4: Detection and Diagnosis of Citrus Huanglongbing in Navel Orange

(19) The embodiment is conducted in the orchard of Atoca Orange Limited located in Lechang, Shaoguan from May 15.sup.th to 17.sup.th in 2013. A dark treatment was performed at 5:00 p.m. on May 15.sup.th (leaves of the navel orange to be detected were bagged with black plastic bags, and then the bags were sealed with clamps). At 6:00 a.m. on May 16.sup.th, the leaves (the leaves to be detected were leaves infected with the citrus huanglongbing, as diagnosed through detection of nucleic acid molecules) were collected as dark-treated leaves and brought indoors (the sampling cite was about 100 M away from indoor). Subsequently the leaves were frozen in the freezer of a refrigerator under a freezing temperature of 4 C. for 24 hours, and then clipped into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), so as to obtain frozen leaves. The frozen leaves were transferred into test tube, and then a chlorophyll decoloring solution (methyl benzene) of 25 ml was added into each test tube, wherein during the addition, the tube was shaken for 2-3 times. After 10 hours, the thin-strip-shaped leaves turned white. Thereafter decolored leaves were obtained by pouring out the decoloring solution and taking the leaves out. Then a starch color-development solution (iodine tincture) was directly added onto the thin strips of the decolored leaves dropwise so as to perform a diagnosis by observing the color change of the leaves.

(20) The result is as shown in FIG. 5, wherein in FIGS. 5, B and C are leaves to be detected in this embodiment, and A is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 5 that after the leaves (B and C) to be detected in this embodiment are color-developed, the thin-strip-shaped leaves turn blue, such that the leaves (B and C) to be detected are determined to be leaves infected with the citrus huanglongbing; and the color of the normal leaf (A) is not changed, such that the leaf (A) is determined to be a normal leaf not infected with the citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(21) Embodiment 5: Detection and Diagnosis of Citrus Huanglongbing in Hongjiang Orange

(22) The embodiment is conducted in Qingping Orchard located in Lianjiang. At 1:00 p.m. on May 23, 2013, a branch of Hongjiang orange to be detected was collected and brought indoors. The branch was inserted in water and then stayed in a dark room. At 8:00 a.m. on May 24.sup.th, leaves were collected from the branch as dark-treated leaves. Subsequently the leaves were frozen in the freezer of a refrigerator under a freezing temperature of 4 C. for 24 hours, and then clipped into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), so as to obtain frozen leaves. The frozen leaves were transferred into test tubes, and then a chlorophyll decoloring solution (methyl benzene) of 25 ml was added into each tube, wherein during the addition, the tube was shaken for 2-3 times. After 6 hours, the thin-strip-shaped leaves turned white. Thereafter the decoloring solution was poured out, and a starch color-development solution (iodine tincture) was added dropwise so as to perform a diagnosis by observing the color change of the leaves.

(23) The result is as shown in FIG. 6, wherein in FIG. 6, A is the leaf to be detected in this embodiment, and B is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 6 that after the leaf (A) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue, such that the leaf (A) to be detected is determined as a leaf infected with the citrus huanglongbing; and the color of the normal leaf (B) is not changed such that the leaf is determined to be a normal leaf not infected with the citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(24) Embodiment 6: Detection and Diagnosis of Citrus Huanglongbing in Summer Orange

(25) The samples are taken from the summer orange orchard in Beihai. A dark treatment was performed in the afternoon on May 25, 2013 (leaves of the summer orange to be detected were bagged with black plastic bags, and then the bags were sealed with clamps). At 7:00 a.m. on May 26.sup.th, the dark-treated leaves (the leaves to be detected were leaves infected with the citrus huanglongbing, as diagnosed through detection of nucleic acid molecules) were collected. Subsequently the leaves were frozen in the freezer of a refrigerator under a freezing temperature of 12 C. The leaves were taken out at 6:00 a.m. on May 27.sup.th and then clipped into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), so as to obtain frozen leaves. The frozen leaves were transferred into test tubes, and then a chlorophyll decoloring solution (methyl benzene) of 25 ml was added into each test tube, wherein during the addition, the tube was shaken for 2-3 times. At twelve in the noon, the thin-strip-shaped leaves turned white. Thereafter a starch color-development solution (iodine tincture) was directly added onto the thin strips of the decolored leaves dropwise, so as to perform a diagnosis by observing the color change of the leaves.

(26) The result is as shown in FIG. 7, wherein in FIG. 7, A is the leaf to be detected in this embodiment, and B is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 7 that after the leaf (A) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue, such that the leaf to be detected is determined to be a leaf infected with the citrus huanglongbing; and the color of the normal leaf (B) is not changed, such that the leaf is determined to be a normal leaf not infected with the citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.

(27) Embodiment 7: Detection and Diagnosis of Citrus Huanglongbing in Citrus Gonggan

(28) A branch of Citrus Gonggan to be detected was collected from the citrus orchard in Deqing on May 29, 2013 and brought indoors. The branch was inserted in water and then its leaves were bagged with black bags for a dark treatment of 24 hours, so as to obtain dark-treated leaves (the leaves to be detected were leaves infected with the citrus huanglongbing, as diagnosed through detection of nucleic acid molecules). Subsequently at 6:00 p.m. on May 30.sup.th, the dark-treated leaves were frozen in the freezer of a refrigerator under a freezing temperature of 10 C. for 14 hours, and then at 8:00 a.m. on May 31.sup.th, the leaves were clipped into thin strips with a width of 1 mm (each leaf was clipped on one side of the main vein from a side edge of the leaf along a leaf lateral vein direction towards the main vein and until the main vein was reached, without cutting off the main vein, so as to clip the leaves into several thin strips with the width of 1 mm, as shown in FIG. 1A), so as to obtain frozen leaves. The frozen leaves were transferred into test tubes, and then a chlorophyll decoloring solution (methyl benzene) of 25 ml was added into each test tube, wherein during the addition, the tube was shaken for 2-3 times. At 3:00 p.m., the thin strips of leaves turned white and thus decolored leaves were obtained. Then a starch color-development solution (iodine tincture) was directly added onto the thin strips of the decolored leaves dropwise, so as to perform a diagnosis by observing the color change of the leaves. The result is as shown in FIG. 8, wherein in FIG. 8, B is the leaf to be detected in this embodiment, and A is a normal leaf not infected with the citrus huanglongbing as proved by the prior art. It can be seen from FIG. 8 that after the leaf (B) to be detected in this embodiment is color-developed, the thin-strip-shaped leaf turns blue, such that the leaf to be detected is determined to be a leaf infected with the citrus huanglongbing; and the color of the normal leaf (A) is not changed, such that the leaf (A) is determined to be a normal leaf not infected with the citrus huanglongbing. This detection result is consistent with a detection result obtained by diagnosing with a conventional detection of nucleic acid molecule.