CN111937622B - Inoculation method of citrus canker pathogen - Google Patents
Inoculation method of citrus canker pathogen Download PDFInfo
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- CN111937622B CN111937622B CN202010831099.5A CN202010831099A CN111937622B CN 111937622 B CN111937622 B CN 111937622B CN 202010831099 A CN202010831099 A CN 202010831099A CN 111937622 B CN111937622 B CN 111937622B
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Abstract
The invention discloses an inoculation method of citrus canker pathogenic bacteria, and belongs to the technical field of citrus canker prevention and control. The inoculation method of citrus canker pathogenic bacteria comprises the following steps: step 1: preparing an inoculation tool; step 2: treatment of citrus leaves infected with canker; and step 3: preparing a test material; and 4, step 4: inoculating; and 5: and (4) treating the inoculated leaves. Compared with the inoculation method in the prior art, the inoculation method of the citrus canker pathogenic bacteria can quickly and efficiently complete the artificial inoculation of the citrus canker pathogenic bacteria, can save a bacterial culture stage, can shorten a test period, greatly improves the test efficiency, can process samples in large batch, and has strong reliability of test data; moreover, the inoculation disease condition is easy to control, can be completed without instruments, and can realize the standardization and the batch production of the method.
Description
Technical Field
The invention relates to an inoculation method of citrus canker pathogenic bacteria, and belongs to the technical field of citrus canker prevention and control.
Background
The orange is worldwideChina is the largest citrus producing and consuming country in the world. The citrus cultivation is widely distributed in more than 20 provinces and cities in the south of China, and the citrus industry is also a main income source of farmers in main producing areas. The Wo citrus as a newly developed late-maturing citrus has the characteristics of vigorous tree vigor, early fruiting, high yield, beautiful fruit appearance, tender pulp, slag melting, multi-flavor and sweet juice, high sugar content, low acid content, easy cultivation and management and the like, and is popular with fruit growers in Guangxi province. The volume of the wild oranges planted in the whole area of Guangxi province at day-ahead is very high, the seeds are quickly expanded, and nearly 7000hm is planted in the short 2-year period 2 Wherein only Nanning has been developed to 2000-3000 hm.
Ulcer is a worldwide disease which occurs in almost every citrus producing country in asia and is distributed in the provinces of guangdong, guangxi, fujian, jiang, zhejiang, jiangsu, hunan, north of lake, Guizhou, Sichuan and the like in china. The method is characterized in that branches and tips of citrus leaves and fruits are damaged, the seedlings and young trees are damaged the most seriously, and the fallen leaves, the withered tips, the weak tree vigor, the fallen fruits and the like are caused, so that the yield and the quality are seriously influenced. The canker can damage dozens of plants in the Rutaceae, and has wide transmission route, rapid infection and serious damage.
Citrus canker pathogen (Xanthomonas subsp. citri, Xcc) is a bacterium belonging to the order Pseudomonas, family Xanthomonas, genus Xanthomonas, and causes canker in citrus. Gram-negative bacteria, aerobic, brevibacterium. The resulting diseases occur mainly on leaves, twigs, thorns, old branches and fruits, causing fruit and leaf drop.
In the inoculation method of citrus canker pathogenic bacteria in the prior art, an adopted inoculation tool generally comprises an insect needle (1) and a dropper (2) (as shown in figure 1), the dropper (2) comprises a suction bag (3) and a suction pipe (4) which are communicated, and a conical suction opening (5) is arranged at one end of the suction pipe (4) far away from the suction bag (3). When inoculating, firstly, the insect needle (1) is used for needling one needle hole at a time; then the pathogenic bacteria liquid is absorbed by the drop (2) and is dropped into the pierced needle hole one by one. The disadvantages of the above described inoculation tools are: the inoculation time is long, and the labor hour is wasted by quantifying the inoculation.
In addition, sand paper is used in the prior art for inoculating the citrus leaves after the citrus leaves are wounded, and the defects that the inoculation area is large or small and cannot be quantified are overcome.
In addition, the inoculation of citrus canker pathogenic bacteria in the prior art also comprises a pure culture process of the pathogenic bacteria on a culture medium, so that the operation is complicated, and the required time is long.
In view of the above, there is a need to provide a new method for inoculating citrus canker pathogen to solve the deficiencies of the prior art.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for inoculating citrus canker pathogens. Compared with the inoculation method in the prior art, the inoculation method of the citrus canker pathogenic bacteria can quickly and efficiently complete the artificial inoculation of the citrus canker pathogenic bacteria, can save a bacterial culture stage, can shorten a test period, greatly improves the test efficiency, can process samples in large batch, and has strong reliability of test data; moreover, the inoculation disease condition is easy to control, can be completed without instruments, and can realize the standardization and the batch production of the method.
The technical scheme for solving the technical problems is as follows: the inoculation method of citrus canker pathogenic bacteria comprises the following steps:
step 1: preparation of inoculating tools
The inoculation tool comprises a tattooing needle sheet and a dropper, wherein the tattooing needle sheet comprises a fixing sheet and a plurality of stainless steel needles fixedly arranged on one side of the fixing sheet; the dropper comprises a suction bag (3) and a suction pipe (4) which are connected in a penetrating way; one end of the fixing piece, which is far away from the stainless steel needles, is arranged at the position of the suction pipe, which is far away from the pipe orifice of the suction bag;
step 2: treatment of citrus leaves infected with canker
Collecting citrus leaves infected with canker, adding water 2-3 times of the weight of the citrus leaves, juicing, filtering, and collecting liquid to obtain pathogenic bacteria liquid;
and step 3: preparation of test materials
Taking tender tips and leaves of seedlings of the citrus nutrition bag as test materials;
and 4, step 4: inoculation of
Taking the inoculation tool in the step 1, sucking the pathogenic bacteria liquid in the step 2 through the suction pipe, then carrying out acupuncture on the test material in the step 3 by using a plurality of stainless steels, and dripping 1-2 drops of the pathogenic bacteria liquid in the suction pipe to the acupuncture position;
and 5: treatment of inoculated leaves
And (4) placing the seedlings of the citrus nutrition bags inoculated in the step (4) into a freshness protection package, preserving moisture for 22-26 h, removing the freshness protection package, transferring to the open air, performing conventional management until the inoculated leaves have citrus canker disease symptoms, and counting the morbidity of citrus canker germs.
The principle of the invention is as follows:
firstly, in the step 1 of the invention, the adopted inoculation tool can not only complete the puncture of multiple needles once, but also simultaneously drip out the pathogenic bacteria liquid, and in addition, the preparation stage of the pathogenic bacteria liquid can be omitted, the inoculation time of pathogenic bacteria can be shortened, the efficiency is improved, and the labor and the time are saved.
Secondly, in the step 4 of the invention, when acupuncture is carried out, a plurality of stainless steel needles on the inoculation tool can cross the veins, the needle head slightly penetrates through the leaf tissues, and then pathogenic bacteria liquid in the suction tube is extruded out simultaneously. Generally, each site is required to be one knife.
Thirdly, in the step 5 of the invention, the nursery stock after moisture preservation is transferred to the open air, and the nursery stock receives sunlight, rain and dew, namely the management is carried out as the same as that of the citrus trees which normally grow in the field. This is also a simulated field ecological environment. The disease symptoms after seedling inoculation are consistent with the disease symptoms of infected ulcer of citrus trees in the field, the identification is easy, the inoculation disease conditions are easy to control, and the standardization of the technical method is easy to realize.
The inoculation method of the citrus canker pathogenic bacteria has the beneficial effects that:
1. the inoculation method of the invention can not only complete multi-needle puncture once, but also drip out the pathogenic bacteria liquid, can save the preparation stage of the pathogenic bacteria liquid, can shorten the inoculation time of pathogenic bacteria, improve the efficiency, save labor and time, and can develop the drug effect comparison test in large batch.
2. The inoculation method disclosed by the invention is simple and feasible, easy to operate, easy to control the disease attack conditions of inoculation, easy to realize the standardization of a technical method, high in inoculation success rate and suitable for large-scale popularization and application.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in step 1, the stainless steel needle is any one of 14 needles, 17 needles and 21 needles.
The adoption of the further beneficial effects is as follows: when the stainless steel needle on the inoculation tool is 14 needles, 1 second is required for completing 14-hole inoculation once. In the prior art, 14 seconds are needed for completing 14-hole inoculation by a single insect needle, and the time for dripping pathogenic bacteria is also 14 seconds, namely 28 seconds in total. Therefore, the inoculation efficiency can be improved by 27 times under the same inoculation task.
When the stainless steel needle on the inoculation tool is 17 needles, the inoculation efficiency can be improved by 33 times under the same inoculation task.
When the stainless steel needle on the inoculation tool is 21 needles, the inoculation efficiency can be improved by 41 times under the same inoculation task. The operator can flexibly make a selection according to the actual situation.
Further, in the step 1, one end of the fixing piece, which is far away from the plurality of stainless steel needles, is fixedly connected with the pipe orifice of the suction pipe, which is far away from the suction bag, through an adhesive tape.
The adoption of the further beneficial effects is as follows: when in operation, the conical suction port part of the suction pipe of the dropper in the prior art can be firstly cut off, then the fixing piece is put into the mouth of the suction pipe, and then the multilayer adhesive tapes are wound, so that the tattooing needle piece can be fixed on the dropper, and the operation is more flexible and convenient.
Further, in the step 2, the citrus canker infected leaves have the following characteristics: yellow or yellowish green oil stain-like spots appear on the leaf back, the two sides of the leaf gradually bulge to form a nearly circular beige disease spot, then the epidermis of the disease part is cracked to be spongy, the bulge is obvious, the surface is rough, grey white or grey brown, then the center of the disease part is sunken and cracks in a volcano mouth shape, fine ring lines are formed, yellow halos are formed around the disease part, and the diameter of the disease spot is 3-5 mm.
The adoption of the further beneficial effects is as follows: according to the appearance change of the leaves, whether the leaves are infected with the citrus canker can be judged.
Further, in step 3, the area of the tender tip blade is specifically as follows: the area of the young tip blade of the sugar orange is (2.4-3.3) cm x (5.2-7.6) cm, and the area of the young tip blade of the Wo citrus is (2.8-4.0) cm x (7.3-10.3) cm.
The adoption of the further beneficial effects is as follows: when the area of the tender tip leaves is the parameter, the inoculation requirement can be met.
Drawings
FIG. 1 is a schematic diagram of a prior art inoculating tool.
FIG. 2 is a schematic diagram of the structure of the inoculating tool of the present invention.
Fig. 3 is an enlarged view of a portion of the part 6 in fig. 2.
In the figure, 1 is an insect needle; 2 is a dropper in the prior art; 3 is a suction bag; 4 is a suction pipe; 5 is a conical suction port; 6, tattoo needle sheets; 7 is a dropper of the invention; 8 is a fixing sheet; 9 is a stainless steel needle; 10 is rubberized fabric.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Example 1
The inoculation method of citrus canker pathogen of the embodiment comprises the following steps:
step 1: preparation of inoculating tools
As shown in fig. 2 and 3, the inoculation tool comprises an embroidery stitch 6 and a dropper 7, wherein the embroidery stitch 6 comprises a fixing piece 8 and 14 stainless steel needles 9 fixedly arranged on one side of the fixing piece 8; the dropper 7 comprises a suction bag 3 and a suction pipe 4 which are communicated; one end of the fixing piece 8, which is far away from the 14 stainless steel needles 9, is arranged at the pipe orifice of the suction pipe 4, which is far away from the suction bag 3, through an adhesive tape 10.
Step 2: treatment of citrus leaves infected with canker
The citrus canker infected leaves have the following characteristics: yellow or yellowish green oil stain-like spots appear on the leaf back, the two sides of the leaf gradually bulge to form a nearly circular beige disease spot, then the epidermis of the disease part is cracked to be spongy, the bulge is obvious, the surface is rough, grey white or grey brown, then the center of the disease part is sunken and cracks in a volcano mouth shape, fine ring lines are formed, yellow halos are formed around the disease part, and the diameter of the disease spot is 3-5 mm.
Collecting citrus leaves infected with canker, adding water 2 times of the weight of the citrus leaves, juicing, filtering, and collecting liquid to obtain the pathogenic bacteria bacterial liquid.
And step 3: preparation of test materials
Taking young shoot leaves of the young seedlings of the sugar orange nutrition bags as test materials, wherein the area of the young shoot leaves of the sugar orange is (2.4-3.3) cm multiplied by (5.2-7.6) cm.
And 4, step 4: inoculation of
And (3) taking the inoculation tool in the step (1), sucking the pathogenic bacteria liquid in the step (2) through the suction pipe (4), then carrying out acupuncture on the material to be tested in the step (3) by using a plurality of stainless steel needles (9), and dripping 1-2 drops of the pathogenic bacteria liquid in the suction pipe (4) to the acupuncture position.
And 5: treatment of inoculated leaves
And (4) putting the seedlings of the sugar orange nutrition bags inoculated in the step (4) into a freshness protection bag, preserving moisture for 22h, removing the freshness protection bag, transferring to the open air, performing conventional management until the inoculated leaves have citrus canker symptoms, and counting the morbidity of citrus canker germs.
The stainless steel needle 9 of this example was 14 needles, and it took 1 second to complete the inoculation of 14 wells at a time. In the prior art, 14 seconds are needed for completing 14-hole inoculation by a single insect needle, and the time for dripping pathogenic bacteria is also 14 seconds, namely 28 seconds in total. Therefore, the inoculation efficiency can be improved by 27 times under the same inoculation task. In addition, the success rate of inoculation was 100% in terms of strains and 75% in terms of leaf amount.
Example 2
The inoculation method of citrus canker pathogen of the embodiment comprises the following steps:
step 1: preparation of inoculating tools
As shown in fig. 2 and 3, the inoculation tool comprises an embroidery stitch 6 and a dropper 7, wherein the embroidery stitch 6 comprises a fixing piece 8 and 17 stainless steel needles 9 fixedly arranged on one side of the fixing piece 8; the dropper 7 comprises a suction bag 3 and a suction pipe 4 which are communicated; one end of the fixing piece 8, which is far away from the 17 stainless steel needles 9, is arranged at the pipe orifice of the suction pipe 4, which is far away from the suction bag 3, through an adhesive tape 10.
Step 2: treatment of citrus leaves infected with canker
The citrus canker infected leaves have the following characteristics: yellow or yellowish green oil stain-like spots appear on the leaf back, the two sides of the leaf gradually bulge to form a nearly circular beige disease spot, then the epidermis of the disease part is cracked to be spongy, the bulge is obvious, the surface is rough, grey white or grey brown, then the center of the disease part is sunken and cracks in a volcano mouth shape, fine ring lines are formed, yellow halos are formed around the disease part, and the diameter of the disease spot is 3-5 mm.
Collecting citrus leaves infected with canker, adding water 2.5 times of the weight of the citrus leaves, juicing, filtering, and collecting liquid to obtain the pathogenic bacteria bacterial liquid.
And step 3: preparation of test materials
Taking the young tip leaves of the Or nutrition bag seedlings as a test material, wherein the area of the young tip leaves of the Or is (2.8-4.0) cm multiplied by (7.3-10.3) cm.
And 4, step 4: inoculation of
And (3) taking the inoculation tool in the step (1), sucking the pathogenic bacteria liquid in the step (2) through the suction pipe (4), then carrying out acupuncture on the material to be tested in the step (3) by using a plurality of stainless steel needles (9), and dripping 1-2 drops of the pathogenic bacteria liquid in the suction pipe (4) to the acupuncture position.
And 5: treatment of inoculated leaves
Putting the Wo citrus nutrition bag nursery stocks inoculated in the step 4 into a freshness protection bag, removing the freshness protection bag after preserving moisture for 24h, transferring to the open air, performing conventional management until the inoculated leaves have citrus canker disease symptoms, and counting the morbidity of citrus canker germs.
The stainless steel needle of this example was 17 needles, and it took 1 second to complete 17-well inoculation at a time. In the prior art, 17 seconds are needed for completing 17-hole inoculation by a single insect needle, and the time for dripping pathogenic bacteria is added for 17 seconds, namely 34 seconds in total. Therefore, the inoculation efficiency can be improved by 33 times under the same inoculation task. In addition, the success rate of inoculation was 100% in terms of strains, and 100% in terms of leaf amount.
Example 3
The inoculation method of citrus canker pathogen of the embodiment comprises the following steps:
step 1: preparation of inoculating tools
As shown in fig. 2 and 3, the inoculation tool comprises an embroidery stitch 6 and a dropper 7, wherein the embroidery stitch 6 comprises a fixing piece 8 and 21 stainless steel needles 9 fixedly arranged on one side of the fixing piece 8; the dropper 7 comprises a suction bag 3 and a suction pipe 4 which are communicated; one end of the fixing piece 8, which is far away from the 21 stainless steel needles 9, is arranged at the pipe orifice of the suction pipe 4, which is far away from the suction bag 3, through an adhesive tape 10.
Step 2: treatment of citrus leaves infected with canker
The citrus canker infected leaves have the following characteristics: yellow or yellowish green oil stain-like spots appear on the leaf back, the two sides of the leaf gradually bulge to form a nearly circular beige disease spot, then the epidermis of the disease part is cracked to be spongy, the bulge is obvious, the surface is rough, grey white or grey brown, then the center of the disease part is sunken and cracks in a volcano mouth shape, fine ring lines are formed, yellow halos are formed around the disease part, and the diameter of the disease spot is 3-5 mm.
Collecting citrus leaves infected with canker, adding water in an amount which is 3 times the weight of the citrus leaves, juicing, filtering, and collecting liquid, namely the pathogenic bacteria liquid.
And step 3: preparation of test materials
Taking young shoot leaves of the young seedlings of the sugar orange nutrition bags as test materials, wherein the area of the young shoot leaves of the sugar orange is (2.4-3.3) cm multiplied by (5.2-7.6) cm.
And 4, step 4: inoculation of
And (3) taking the inoculation tool in the step (1), sucking the pathogenic bacteria liquid in the step (2) through the suction pipe (4), then carrying out acupuncture on the material to be tested in the step (3) by using a plurality of stainless steel needles (9), and dripping 1-2 drops of the pathogenic bacteria liquid in the suction pipe (4) to the acupuncture position.
And 5: treatment of inoculated leaves
And (4) putting the seedlings of the sugar orange nutrition bags inoculated in the step (4) into a freshness protection bag, preserving moisture for 26 hours, removing the freshness protection bag, transferring to the open air, performing conventional management until the inoculated leaves have citrus canker disease symptoms, and counting the morbidity of citrus canker bacteria.
The stainless steel needle of this example was 21 needles, and it took 1 second to complete 21-well inoculation at a time. In the prior art, a single insect needle completes 21-hole inoculation, which requires 21 seconds, and the time for dripping pathogenic bacteria also requires 21 seconds, which is 42 seconds in total. Therefore, the inoculation efficiency can be improved by 41 times under the same inoculation task. In addition, the success rate of inoculation was 100% in terms of strains and 85% in terms of leaf amount.
In conclusion, compared with the inoculation method in the prior art, the inoculation method for citrus canker pathogens can quickly and efficiently complete the artificial inoculation of the citrus canker pathogens, can save the bacterial culture stage, can shorten the test period, greatly improves the test efficiency, can process samples in large batch, and has strong test data reliability; moreover, the inoculation disease condition is easy to control, can be completed without instruments, can realize the standardization and the batch of the method, has high inoculation success rate, and is suitable for large-scale popularization and application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (2)
1. The inoculation method of citrus canker pathogenic bacteria is characterized by comprising the following steps:
step 1: preparation of inoculating tools
The inoculation tool comprises a tattooing needle sheet (6) and a dropper (7), wherein the tattooing needle sheet (6) comprises a fixing piece (8) and a plurality of stainless steel needles (9) fixedly arranged on one side of the fixing piece (8); the dropper (7) comprises a suction bag (3) and a suction pipe (4) which are communicated; one end of the fixing piece (8) far away from the stainless steel needles (9) is arranged at a pipe orifice of the suction pipe (4) far away from the suction bag (3); one end of the fixing piece (8) far away from the stainless steel needles (9) is fixedly connected with the pipe orifice of the suction pipe (4) far away from the suction bag (3) through an adhesive tape (10); the stainless steel needle (9) is any one of 14 needles, 17 needles and 21 needles;
step 2: treatment of citrus leaves infected with canker
Collecting citrus leaves infected with canker, adding water 2-3 times of the weight of the citrus leaves, juicing, filtering, and collecting liquid to obtain pathogenic bacteria liquid;
and step 3: preparation of test materials
Taking tender tips and leaves of seedlings of the citrus nutrition bag as test materials; the area of the tender tip blade is specifically as follows: the area of the young shoot blade of the sugar orange is (2.4-3.3) cm multiplied by (5.2-7.6) cm, and the area of the young shoot blade of the wogonian orange is (2.8-4.0) cm multiplied by (7.3-10.3) cm;
and 4, step 4: inoculation of
Taking the inoculation tool in the step 1, sucking the pathogenic bacterium liquid in the step 2 through the suction pipe (4), then carrying out acupuncture on the test material in the step 3 by using a plurality of stainless steel needles (9), and dripping 1-2 drops of the pathogenic bacterium liquid in the suction pipe (4) to the acupuncture position;
and 5: treatment of inoculated leaves
And (4) placing the seedlings of the citrus nutrition bags inoculated in the step (4) into a freshness protection package, preserving moisture for 22-26 h, removing the freshness protection package, transferring to the open air, performing conventional management until the inoculated leaves have citrus canker disease symptoms, and counting the morbidity of citrus canker germs.
2. A method of inoculating citrus canker pathogen as claimed in claim 1 wherein in step 2, the citrus canker infected leaves have the following characteristics: yellow or yellowish green oil stain-like spots appear on the leaf back, the two sides of the leaf gradually bulge to form a nearly circular beige disease spot, then the epidermis of the disease part is cracked to be spongy, the bulge is obvious, the surface is rough, grey white or grey brown, then the center of the disease part is sunken and cracks in a volcano mouth shape, fine ring lines are formed, yellow halos are formed around the disease part, and the diameter of the disease spot is 3-5 mm.
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