阅读说明：本技术 一种调查田间秸秆残茬上禾谷镰刀菌子囊壳数量的方法 (Method for investigating quantity of fusarium graminearum ascochyta on field straw stubble ) 是由 茹艳艳 郝俊杰 谢淑娜 刘佳中 李保叶 孙静 王新涛 于 2021-09-24 设计创作，主要内容包括：本发明提供一种科学准确的调查田间秸秆残茬上禾谷镰刀菌子囊壳数量的方法,将调查方法科学标准化。通过收集的田间秸秆残茬,首先估算产子囊壳面积占整个秸秆表面积的比例,然后在标尺背景下对秸秆上着生子囊壳有代表性的位置进行拍照,利用Image J软件计数照片秸秆上每平方厘米的子囊壳数量,最后换算为每块地平均每平方厘米秸秆产生的子囊壳数量(个/cm～(2))。经过多年多点的调查,建立了一种针对秸秆残茬上禾谷镰刀菌产生子囊壳数量的标准、科学的调查方法,能较准确地反映该地块相关病害的初始菌源量。本方法将调查方法标准统一化,方便基层农技人员操作。(The invention provides a method for scientifically and accurately investigating the quantity of fusarium graminearum cysts on field straw stubbles, and the investigation method is scientifically standardized. Through collected field straw stubble, firstly estimating the proportion of the area of the produced ascocarp to the whole straw surface area, then taking a picture of a representative position of the ascocarp growing on the straw under the background of a scale, counting the number of the ascocarp per square centimeter on the straw by using Image J software, and finally converting the number of the ascocarp generated per square centimeter of the straw per block (number/cm) 2 ). Through years of multi-point investigation, a standard and scientific investigation method for the quantity of ascochyta generated by fusarium graminearum on straw stubbles is established, and the initial bacterial source quantity of diseases related to the plot can be accurately reflected. The method unifies the standard of the investigation method, and is convenient for the operation of the grassroots farmer.)

1. A method for investigating the quantity of fusarium graminearum cysts on field straw stubble is characterized by comprising the following steps:

(1) collecting a certain amount of straw stubble in a field to be investigated;

(2) estimating the proportion of the area of the seed capsule shell produced on each straw to the whole straw surface area;

(3) photographing the straws under the background of a scale;

(4) counting the number of the ascochyta shells on the straws of the photos by using Image J software;

(5) calculating the number of the ascochyta shells generated by the straws per square centimeter in each land;

(6) and determining the initial bacterial source quantity of the related diseases of the area around the field according to the quantity of the ascochyta generated by each square centimeter of straws on average.

2. The method of claim 1, wherein the specific method of step (1) is: and (3) collecting corn straw stubbles or rice straw stubbles on the ground surface of the wheat field at the beginning of 4 months in each year, collecting the wheat straw stubbles on the ground surface of the wheat field at the bottom of 7 months, and randomly collecting 30 straw stubbles with consistent lengths in each field.

3. The method of claim 1, wherein the specific method of step (2) is: observing the size and morphological distribution characteristics of the ascocarp generated on each straw stubble, and estimating the proportion of the ascocarp generated on each straw stubble to the whole straw surface area according to the characteristics, wherein the proportion is represented by p.

4. The method of claim 1, wherein the specific method of step (3) is: taking a picture of a representative position of a raw seed capsule shell on each straw by taking square lattice paper as a scale, wherein the area of each square lattice is a; representative positions therein refer to: can represent the formation condition of the ascocarp on the straw and can clearly count the position of the ascocarp.

5. The method of claim 1, wherein the specific method of step (4) is: running Image J software, clicking "open" in a "file" pull-down menu, opening a straw photo of an ascope shell to be counted, then clicking a selection box representing a square, firstly selecting a square grid in a winning scale in the photo, clicking "Measure" in an "Analyze" pull-down menu, and displaying the area b of the grid below the focal length of the photo, which is calculated by the software, in "Results"; then selecting a representative part of the sub-capsule shells in the straw photo, using a square selection frame to circle a certain area, counting the number of the sub-capsule shells in the area of the frame as n, clicking 'Measure' in an 'Analyze' pull-down menu and 'Results' in display software to calculate and shoot the area c of the circled frame under the focal length of the photo; repeating the steps of calculating n and c for 3 times.

6. The method of claim 1, wherein the specific method of step (5) is: calculating the actual number N (number/cm) of the ascomycetes shells per square centimeter on the surface of each straw according to the principle that the straws and the square lattice background scale are under the same focal length on the picture, and the ratio of the actual area of the scale to the actual area of the straws is equal to the ratio of the area under the focal length²)，N＝[n₁/(c₁/b×a)+n₂/(c₂/b×a)+n₃/(c₃/b×a)]/3 × p, where n₁、n₂、n₃And c₁、c₂、c₃Respectively representing the number of the ascomycetes shells in the circled square area counted by the artificial naked eyes and the circled square area under the focal length of the picture which are repeated for 3 times, and further calculating the average number of the ascomycetes shells generated on each square centimeter of the straws on each land, namely the average value of the number of the ascomycetes shells on 30 straws.

7. The method of claim 1, wherein the number of cysts in step (6) is related to the initial field source of bacteria by:

number of ascochyta (number/cm)²) Initial bacterial source amount ≤1.5 Is extremely deficient 1.6～5.0 Lack of supply 5.1～10.0 Is rich in 10.1～30.0 Rich and sufficient bacteria source ≥30.1 Rich and abundant bacteria source

Technical Field

The invention relates to a method for investigating the quantity of fusarium graminearum ascochyta on field straw stubble, and belongs to the field of investigation of initial bacterial source quantity of crop diseases.

Background

Fusarium graminearum is an ascomycete of Hypocrea (Hypocrea), the asexual generation name being Fusarium graminearum Schwabe, and the sexual generation being Gibberella zeae. Fusarium graminearum is distributed all over the world and mainly infects cereal crops such as barley, wheat, oat, corn, rice and the like and non-gramineous plants such as cotton, beans and the like to cause the occurrence of root rot, stem rot and ear rot of various crops, thereby not only causing the serious yield reduction of the crops and causing huge economic loss, but also causing grain pollution because various fungal toxins produced are remained in the crops and seriously threatening the food safety and the health of people and livestock.

Fusarium graminearum infects crops to cause diseases, and two spore types can be generated during overwintering on crop stubbles: one is asexual conidium, is suitable for being spread by rainwater splash, can be infected and expanded to the ear by wheat leaves, and has limited spreading distance; the other is sexual ascospore, which is sprayed into air from mature ascospore in warm and humid weather condition, and spread by air and rain for a long distance. Fusarium graminearum overwintering on plant residues such as corn, rice and the like in the form of ascocarp, proper climatic conditions are provided for development and formation of the ascocarp due to temperature rise and humidity increase in the spring of the next year, ascospores are ejected out of the ascocarp by virtue of huge swelling pressure generated by water absorption of an ascocarp in the mature ascocarp, and the ascospores are spread in the field by virtue of wind power, and a part of the ascocarp falls on the wheat head. While the conidia of fusarium graminearum can also infect wheat ears after germination, the ascospores are the primary source of infection of wheat scab in the field.

Different from the planting system of one cropping in Europe and America, the yellow-Huaihai wheat-corn crop rotation and the Yangtze river basin wheat-rice crop rotation of two cropping in one year are the special cultivation systems in China. Wherein, the crop rotation mode in southern areas is wheat-rice, so the dormant ascospores on rice stakes and ascospores released in the next year are the main initial infection sources of wheat scab; the crop rotation mode in northern areas is wheat-corn, so the ascospores released in the next year and ascospores on the residual stubble of corn on the surface of the field are the main initial infection sources of wheat scab; on the stubble after wheat harvest, fusarium graminearum can also form an ascocarp, and provides a bacterial source for stem rot and ear rot of corn, damping-off of rice and the like of a next-stubble crop. Therefore, the accurate judgment of the initial field bacterial source quantity can provide important basis for the prediction and forecast of the related diseases.

The previous judgment of the initial bacterial source amount in the field is mainly expressed by investigating the bacterial carrying rate of the straw stubbles in the field, and the investigation method of the quantity of the ascochyta on the straws comprises the steps of bringing the collected straw stubbles back to the room, using a dissecting microscope, selecting 5-8 points before counting for each straw, wherein the area of each point is 0.1923cm²Counting the number of the ascocarp in the selected area under 30 or 40 times of a dissecting microscope, and then converting the number of the ascocarp into the number of the ascocarp per square centimeter on the average of the straw surface area. This method is cumbersome and time consuming, and requires the operator to have the expertise to use the dissecting microscope. Therefore, a simpler, time-saving and accurate method is needed for judging the number of ascochyta on straw stubbles and providing more accurate initial bacteria source amount data for prediction and forecast of related diseases.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for scientifically and accurately investigating the quantity of fusarium graminearum ascochyta on field straw stubbles.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for investigating the quantity of fusarium graminearum ascochyta on field straw stubble comprises the following steps:

(1) collecting a certain amount of straw stubble in a field to be investigated;

(2) estimating the proportion of the area of the seed capsule shell produced on each straw to the whole straw surface area;

(3) photographing the straws under the background of a scale;

(4) counting the number of the ascochyta shells on the straws of the photos by using Image J software;

(5) calculating the number of the ascochyta shells generated by the straws per square centimeter in each land;

(6) and determining the initial bacterial source quantity of the related diseases of the area around the field according to the quantity of the ascochyta generated by each square centimeter of straws on average.

The specific method of the step (1) is as follows: and (3) collecting corn straw stubbles or rice straw stubbles on the ground surface of the wheat field at the beginning of 4 months in each year, collecting the wheat straw stubbles on the ground surface of the wheat field at the bottom of 7 months, and randomly collecting 30 straw stubbles with consistent lengths in each field.

The specific method of the step (2) is as follows: observing the size and morphological distribution characteristics of the ascocarp generated on each straw stubble, and estimating the proportion of the ascocarp generated on each straw stubble to the whole straw surface area according to the characteristics, wherein the proportion is represented by p.

The specific method of the step (3) is as follows: taking a picture of a representative position of a raw seed capsule shell on each straw by taking square lattice paper as a scale, wherein the area of each square lattice is a; representative positions therein refer to: can represent the formation condition of the ascocarp on the straw and can clearly count the position of the ascocarp.

The specific method of the step (4) is as follows: running Image J software, clicking "open" in a "file" pull-down menu, opening a straw photo of an ascope shell to be counted, then clicking a selection box representing a square, firstly selecting a square grid in a winning scale in the photo, clicking "Measure" in an "Analyze" pull-down menu, and displaying the area b of the grid below the focal length of the photo, which is calculated by the software, in "Results"; then selecting a representative part of the sub-capsule shells in the straw photo, using a square selection frame to circle a certain area, counting the number of the sub-capsule shells in the area of the frame as n, clicking 'Measure' in an 'Analyze' pull-down menu and 'Results' in display software to calculate and shoot the area c of the circled frame under the focal length of the photo; repeating the steps of calculating n and c for 3 times.

The specific method of the step (5) is as follows: calculating the actual number N (number/cm) of the ascomycetes shells per square centimeter on the surface of each straw according to the principle that the straws and the square lattice background scale are under the same focal length on the picture, and the ratio of the actual area of the scale to the actual area of the straws is equal to the ratio of the area under the focal length²)，N＝[n₁/(c₁/b×a)+n₂/(c₂/b×a)+n₃/(c₃/b×a)]/3 × p, where n₁、n₂、n₃And c₁、c₂、c₃Respectively representing the number of the ascomycetes shells in the circled square area counted by the artificial naked eyes and the circled square area under the focal length of the picture which are repeated for 3 times, and further calculating the average number of the ascomycetes shells generated on each square centimeter of the straws on each land, namely the average value of the number of the ascomycetes shells on 30 straws.

The relation between the quantity of the ascochyta in the step (6) and the initial field bacterial source quantity is as follows:

number of ascochyta (number/cm)2)	Initial bacterial source amount
		≤1.5	Is extremely deficient
1.6～5.0	Lack of supply
		5.1～10.0	Is rich in
10.1～30.0	Rich and sufficient bacteria source
		≥30.1	Rich and abundant bacteria source

The invention has the beneficial effects that:

1. the application surveys the number of ascospores generated by fusarium graminearum on field straw stubbles. The method is scientific and accurate in the quantity investigation of the ascochyta shells generated by fusarium graminearum on the field straw stubbles under the wheat-corn or wheat-rice rotation mode, the field straw stubbles are collected at the beginning of 4 months and at the end of 7 months per year, the quantity of the generated ascochyta shells can be investigated more accurately, and more accurate initial bacteria source quantity data is provided for prediction and forecast of related diseases.

2. The application scientifically standardizes the investigation method. Through collected field straw stubble, firstly estimating the proportion of the area of the produced ascocarp to the whole straw surface area, then taking a picture of a representative position of the ascocarp growing on the straw under the background of a scale, counting the number of the ascocarp per square centimeter on the straw by using Image J software, and finally converting the number of the ascocarp generated per square centimeter of the straw per block (number/cm)²). Through years of multi-point investigation, a standard and scientific investigation method for the quantity of ascochyta generated by fusarium graminearum on straw stubbles is established, and the initial bacterial source quantity of diseases related to the plot can be accurately reflected. The method unifies the standard of the investigation method, and is convenient for the operation of the grassroots farmer.

Drawings

FIG. 1 is a schematic diagram of a method for investigating the number of ascochyta on a single straw stubble in example 1.

FIG. 2 is a schematic diagram of a method for investigating the number of ascochyta on a single straw stubble in example 2.

Wherein, in FIGS. 1 and 2, a is a solid square lattice of a scaleArea (0.7cm × 0.7cm ═ 0.49 cm)²) B is the area of the grid below the focal length of the photo, which is calculated by software, c1, c2 and c3 are the area of the circled boxes under the focal length of the photo, and n1, n2 and n3 are the numbers of the sub-capsule shells in the circled box areas, which are counted by the naked human eyes.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Example 1

A method for investigating the quantity of fusarium graminearum ascochyta on field straw stubble, which is carried out in four areas of Rooshan county, Tanmaji county, Changchang Kudzuvine county and Yuanyang county of Xinyang city in Henan province, at the beginning of 4 months and at the end of 7 months in 2019, and comprises the following steps:

(1) collection of field straw stubble

Collecting rice straw stubble on the ground surface in a wheat field of Xinyang Luoshan about 4 months and 5 days, and collecting corn straw stubble on the ground surface in a wheat field of Xinyang Luoshan, Xinyang Changgu and Xinxiangyuan Yuanyang; collecting wheat straw stubble on the ground surface in about 7 months and 30 days, and randomly collecting 30 straw stubble with consistent length in each land (because the straws on the ground surface in the field are different in length and different in bacteria carrying amount, the number of the ascomycetes on some straws is large, and the number of the ascomycetes on some straws is small, if the number of the collected straws is too small, the actual situation of the whole land cannot be represented, and if the number of the collected straws is too large, the workload is large, so that 30 times of repeated sampling are carried out, and finally, the calculated average number of the ascomycetes generated by the straws per square centimeter in each land is the average value of 30 times);

(2) estimating the ratio of the area of the ascope shell on each straw to the whole straw surface area

Observing the size and morphological distribution characteristics (whether the distribution of the ascocarp shells is uniform, the density, the size of the ascocarp shell stack and the like) of the ascocarp shells generated on each straw stubble, and estimating the proportion of the area of the ascocarp shells generated on each straw stubble to the surface area of the whole straw according to the characteristics, wherein the proportion is represented by p;

(3) shoot the straw under the background of the scale

Use methodGrid (area a cm)²Long cm multiplied by width cm) paper is taken as a scale, and representative positions of the seed capsule shells growing on each straw are photographed under a camera macro lens (only the seed capsule shells on the straws can be clearly photographed); representative positions therein refer to: can represent the formation condition of the ascocarp on the straw and can clearly count the position of the ascocarp;

(4) counting the number of ascochyta on the straw of the photo by using Image J software

Downloading Image J installation software (ij143-jdk6-setup. exe) on the internet, operating the software to click "open" in a "file" pull-down menu after successful installation on a computer according to a prompt, opening a straw photo needing counting of an ascope shell, then clicking a selection frame representing a square, firstly selecting a square grid in a winning scale in the photo, clicking "Measure" in an "Analyze" pull-down menu, and displaying an area b for shooting the grid below the focal length of the photo calculated by the software in "Results"; then selecting a representative position of the growing ascocarp shell in the straw photo, using a square selection frame to circle a certain area, counting the number of the ascocarp shells in the area of the frame as n by naked eyes, clicking 'Measure' in an 'Analyze' pull-down menu, and displaying the area c of the circled frame under the focal length of the photo calculated by software in 'Results'; repeating the steps of calculating n and c for 3 times;

(5) calculating the average per square centimeter straw per plot

Calculating the actual number N (number/cm) of the ascomycetes shells per square centimeter on the surface of each straw according to the principle that the straws and the square lattice background scale are under the same focal length on the picture, and the ratio of the actual area of the scale to the actual area of the straws is equal to the ratio of the area under the focal length²)，N＝[n₁/(c₁/b×a)+n₂/(c₂/b×a)+n₃/(c₃/b×a)]/3 × p, where n₁、c₁、n₂And c₂、n₃、c₃Respectively representing the number of the ascomycetes shells in the circled square area counted by the artificial naked eye and the circled square area under the focal length of the picture which are repeated for 3 times, and further calculating the number of the ascomycetes shells generated by the straw per square centimeter of each land, namelyAverage value of number of ascocarp on 30 straws.

(6) Determination of initial bacterial origin

The initial bacterial source quantity of the diseases related to the area around the field is determined according to the average number of the ascochyta generated by straws per square centimeter of each field, and the division standard of the initial bacterial source quantity is shown in a table 1.

TABLE 1 determination of initial bacterial source in the field

Number of ascochyta (number/cm)2)	Initial bacterial source amount
		≤1.5	Is extremely deficient
1.6～5.0	Lack of supply
		5.1～10.0	Is rich in
10.1～30.0	Rich and sufficient bacteria source
		≥30.1	Rich and abundant bacteria source

The number of the ascochyta shells on the straw residues on the ground surface collected in the 4 places is investigated, and the specific calculation process is shown in table 2 and fig. 1 (the calculation process of the number of the ascochyta shells on a single straw residue is only listed as an exhibition). Compared with the number of the ascochyta on the surface straw investigated by the traditional anatomical lens method, the error rate of the investigation data of the method is not more than 5%.

30 straw stubble residues are collected in each place, and finally, the number of the ascochyta generated by the straw per square centimeter in each place is calculated. The survey result shows that: the average number of ascochyta of rice straw stubble on Xinyang Rooshan wheat field surface in the early 4 months in 2019 is 4.5 per cm²The average numbers of the ascospores on the corn straw stubble on the fields of the Shuping wheat, the Changgu wheat and the Yuanyang wheat are respectively 2.6, 1.8 and 1.3/cm²The straw stubble on the wheat field surface of 4 places generates less ascochyta, and the initial infection source of wheat scab is deficient; the average number of the ascospores on the wheat straws on the ground surfaces of the fields of the Rooshan, the Tuodiping, the Changgu and the Yuanyang at the bottom of 7 months is 28.3, 18.6, 13.8 and 10.4 per cm respectively²And the number of the ascospores generated on the wheat straws on the surface of 4 places is large, and the initial bacterial sources of the diseases related to corn stalk rot, ear rot, rice damping-off and the like are very rich (Table 3).

TABLE 2 survey of the number of ascochyta on individual straw stubble

Wherein v is the area calculated by software, and v ═ b or c1, c2 and c3 of square grids or straws; w is the actual area, and the square lattice w is 0.7cm × 0.7cm or 0.49cm²And the straw w is c1/b a, c2/b a and c3/b a.

Taking corn stalks as an example, w1 ═ c1/b ═ a ═ 71968/90552 ═ 0.49 ═ 0.3894, w2 ═ c2/b ═ a ═ 99680/90552 ═ 0.49 ═ 0.5394, w3 ═ c3/b ═ a ═ 109880/90552 ═ 0.49 ═ 0.5946, x is the number of subcapsules, wherein n1 ═ 13, n2 ═ 18, and n3 ═ 16; y is the number of the straw surface per square centimeter of the seed capsule, wherein y1, n1, w1, 13, 0.3894, 33.38, y2, n2, w2, 18, 0.5394, 33.37, y3, n3, w3, 16, 0.5945, 26.91; z is the average number of the ascomycetes shells per square centimeter on the surface of the straw, and z is (y1+ y2+ y3)/3 is (33.38+33.37+26.91)/3 is 31.22; p is the proportion of the area of the ascocarp shell in the surface area of the whole straw, and p is 7.5 percent; n is the average number of the ascocarp per square centimeter of the actual straw surface, and N is 31.22 is 7.5 is 2.3415.

In the dissecting mirror survey method, R is the total number of subcapsular shells of 8 points, and the area of each point is 0.1923cm²M is the number of ascocarp on the straw, and M is (R1+ R2+ R3)/3/(0.1923 × 8) (3+4+4)/3/(0.1923 × 8) ═ 2.3834.

S is the error rate, (N-M)/M100% (2.3415-2.3834)/2.3834 100% (-1.76%).

Table 32019 survey results of average numbers of ascochyta on straw stubble on surface of 4 sites in year

Example 2

In the beginning of 4 months and at the end of 7 months in 2020, the same method as in example 1 is adopted, and the straw stubble is collected in the plots of Xinyang Luoshan, Lima shop, xuchang Changgu and Xinxiang Yunyang, and the number of ascocarp shells on the straw stubble is investigated, and the specific method and steps are the same as in example 1.

The number of the ascochyta shells on the straw residues on the ground surface collected in the 4 places is investigated, and the specific calculation process is shown in table 4 and fig. 2 (the calculation process of the number of the ascochyta shells on a single straw residue is only listed as an exhibition). Compared with the traditional anatomical lens method for investigating the number of the ascochyta on the straw on the ground surface, the error rate of the investigation data of the method is not more than 3%.

30 straw stubble residues are collected in each place, and finally, the number of the ascochyta generated by the straw per square centimeter in each place is calculated. The results show that: more ascochyta is generated on rice straws and corn straw stubbles on the surfaces of wheat fields in 4 places in the early 4 months of 2020, and the average number of ascochyta is 10.3-27.9/cm²The initial infection sources of wheat scab are very rich; the average number of the ascospores on the wheat straw on the ground surface of the field of the Rooshan, Tuodiping, Changgu and Yuanyang is 2.2, 1.7, 1.5 and 1.2/cm at the bottom of 7 months²In 4 places, the number of the ascospores on the wheat straw is small, and the initial bacterial sources of diseases related to corn stalk rot, corn ear rot, rice damping-off and the like are short (Table 5).

TABLE 4 investigation results of the number of ascochyta on a single straw stubble

Wherein v is the area calculated by software, and v ═ b or c1, c2 and c3 of square grids or straws; w is the actual area, and the square lattice w is 0.7cm × 0.7cm or 0.49cm²And the straw w is c1/b a, c2/b a and c3/b a.

Taking rice straws as an example, w1 ═ c1/b ═ a ═ 76024/246960 ═ 0.49 ═ 0.1508, w2 ═ c2/b ═ a ═ 113904/246960 ═ 0.49 ═ 0.2260, w3 ═ c3/b ═ a ═ 136136/246960 ═ 0.49 ═ 0.2701, x is the number of sub-capsules, wherein n1 ═ 11, n2 ═ 17, and n3 ═ 17; y is the number of the straw surface per square centimeter of the seed capsule, wherein y1, n1, w1, 11, 0.1508, 72.94, y2, n2, w2, 17, 0.2260, 75.22, y3, n3, w3, 17, 0.2701, 62.94; z is the average number of the seed capsule shells per square centimeter of the surface of the straw, and z is (y1+ y2+ y3)/3 is (72.94+75.22+62.94)/3 is 70.37; p is the proportion of the area of the ascocarp shell in the surface area of the whole straw, and p is 40.0 percent; n is the average number of the seed capsule shells per square centimeter of the actual straw surface, and N is 70.37, 40.0 and 28.1480.

In the dissecting mirror survey method, R is the total number of subcapsular shells of 8 points, and the area of each point is 0.1923cm²M is the number of ascocarp on the straw, and M is (R1+ R2+ R3)/3/(0.1923 × 8) (48+43+42)/3/(0.1923 × 8) ═ 28.8178. S is the error rate, (N-M)/M100% (28.1480-28.8178)/28.8178 100% (-2.32%).

Table 52020 survey results of average numbers of ascochyta on straw stubble on surface of 4 sites in 20 years