CN113564076A - Indicator strain and method for predicting rice bacterial leaf streak occurrence period by using bacteriophage - Google Patents
Indicator strain and method for predicting rice bacterial leaf streak occurrence period by using bacteriophage Download PDFInfo
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Abstract
The invention discloses an indicator strain and a method for predicting the emergence period of rice bacterial leaf streak by using phage, and belongs to the technical field of plant protection. According to the method, the rice bacterial leaf streak strain XZ45 with stable biological characteristics, strong pathogenicity and wide affinity is selected as a phage indicator strain, and the phage rapid-increase period is set to be not less than 150 pfu/mL. The method comprises the following steps: collecting a paddy field water or mud sample, and filtering and sterilizing by using a bacterial filter; preparing phage indicator bacterium liquid; uniformly mixing the sample sterile filtrate and the phage indicator bacterium solution, adding a low-temperature liquid NA culture medium, uniformly mixing, culturing for 8-16h at 26-30 ℃ after solidification, and investigating phage density; and (3) when the density of the phage reaches or exceeds 150pfu/mL, entering a sudden increase period, and predicting the occurrence period of the rice bacterial leaf streak by combining medium and short term weather forecast and the rice growth period.
Description
Technical Field
The invention belongs to the technical field of plant protection, and particularly relates to an indicator strain and a technical method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage.
Background
The bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola is one of the important diseases in rice production in China, belongs to the epidemic disease detection of plants in China, and has the annual harmful area of 300 plus 500 mu, mainly causes the loss of 5-15 percent of rice yield, and seriously reaches more than 50 percent. The bacterial leaf streak of the rice mainly overwinter on paddy and diseased straw harvested in a diseased field and becomes a main initial infection source in the second year; after the rice is sowed or transplanted, the bacterial leaf streak germs of the rice start to slowly proliferate in the field, the pathogenic bacteria finish infection and rapidly proliferate under proper conditions to trigger the bacterial leaf streak of the rice, and then the bacterial leaf streak of the rice is spread in the field by means of the exposure of wind and rain to cause serious harm. Currently, the main measures for preventing and controlling the bacterial leaf streak of rice are prevention, and registered medicaments such as thiediazole copper, benziothiazolinone, zinc thiazole and the like can obtain good prevention and control effects when used at the early stage of disease attack, but the treatment effect on the diseases is general. Therefore, accurate prediction of the disease occurrence period provides technical support for prevention and control of bacterial leaf streak of rice. At present, a field is adopted to periodically carry out investigation or establish a special prediction garden to investigate an initial disease point or a disease center, and then medium-term and short-term weather forecast is combined to predict the occurrence and development of diseases. The existing method can only issue early warning information after a disease occurs, and cannot accurately predict the disease occurrence period, so that the prevention and control work of the rice bacterial leaf streak in the field shows hysteresis, and the overall prevention and control effect is not high.
There is a high degree of specificity between phage and host bacteria, and the number of phage is positively correlated with the number of host bacteria. The phage can quickly crack host bacteria, and form macroscopic transparent plaques on a culture medium solid plate containing the host bacteria, and the quantity of the host bacteria can be quickly and simply evaluated through the formation quantity of the plaques. During the period from the infection of rice by bacterial leaf streak germs to the manifestation of disease symptoms, pathogenic bacteria rapidly proliferate, and the number of phages in rice field water also rapidly increases correspondingly (rapid increase period); by monitoring the number of phages in the field water, the occurrence time of the bacterial leaf streak of rice can be predicted, so that the disease prevention and control can be carried out in due time, the disease prevention and control effect can be improved, and the dosage of chemical pesticides can be reduced.
However, because the affinity between the phage and the bacterial strains is differentiated, and the development of the rice bacterial leaf streak is also influenced by internal and external factors, the technical method for predicting the emergence period of the rice bacterial leaf streak by using the phage is immature, the prediction accuracy is low, and the method lacks practical guiding value for disease prevention and control. Therefore, the method for predicting the occurrence period of the bacterial leaf streak of the rice is rapid, simple and accurate, and has important guiding value for preventing and controlling diseases.
Disclosure of Invention
The invention aims to provide an indicator strain and a technical method for predicting the emergence period of rice bacterial leaf streak by using bacteriophage, so as to solve the technical problems, and enable the emergence period of the rice bacterial leaf streak to be rapidly, simply and accurately predicted by using the bacteriophage.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the indicator strain is numbered as XZ45, and is identified as Xanthomonas oryzae pathogenic strain (Xanthomonas oryzae pv. oryzicola) by combining colony morphology observation, thallus morphology observation and 16S rRNA gene sequence determination. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3, 8 and 2021, and the preservation number of the strain is CGMCC No. 21877. The indicator strain has the characteristics of sensitivity and wide affinity, and can predict and indicate the occurrence of the rice bacterial leaf streak with higher sensitivity.
The bacterial colony characteristics of the rice bacterial streak germ XZ45 after being cultured for 36-48h on a NA solid culture medium plate under the condition of 28 ℃: the bacterial colony is smooth, opaque, glossy, round, convex and complete in edge; initially white and then light yellow to a diameter of 1-1.5 mm. The shape of the cells observed by a transmission electron microscope: the thallus is short rod-shaped, and the size is (0.4-0.6) mu m multiplied by (1.1-2.0) mu m; no spore and capsule, and extracellular polysaccharide with mucilage outside the thalli; single generation, few pairs and no chain shape; one polar flagellum (fig. 1).
The invention provides a method for predicting the emergence period of rice bacterial leaf streak by using bacteriophage, which comprises the following steps:
(1) sample collection and preparation: collecting field water for 1 time every 3-10 days from the time of transplanting and turning green or the tillering stage of direct-seeding rice, and filtering and sterilizing to form a sterile sample; if the field is anhydrous, collecting field mud for replacement, adding sterile water with the weight 2-3 times of that of the field mud, uniformly mixing, standing, taking supernatant, and filtering for sterilization to form a sterile sample;
(2) preparing an indication bacterial liquid: inoculating the indicator strain XZ45 into NB liquid culture medium, culturing at 28-30 deg.C for 36-48h, shaking at 120-200 r/min, and adjusting thallus concentration to 3.0 × 10 with sterile water8-2.0×109cfu/mL as an indicator bacterium liquid;
(3) and (3) phage detection: adding 0.1-2.0mL of the sterile sample in the step (1) and 0.5-2mL of the indication bacterial liquid in the step (2) into a sterile culture dish, uniformly mixing, then adding a liquid NA culture medium at 40-45 ℃, rapidly and uniformly mixing, performing inverted culture for 10-16h at 28-30 ℃ after solidification, investigating the number of plaques, and counting the density of phage in field water or mud;
(4) prediction of the emergence period of rice bacterial leaf streak: when the rice is in a tillering stage, when the density of phage is more than or equal to 150pfu/mL, the day with the highest temperature of 25-35 ℃ in 7-14 days in the future is more than or equal to 70%, the number of rainfall days is more than or equal to 40%, and the rice bacterial leaf streak of the field is predicted to occur in 7-14 days in the future; when the rice is in the booting stage, when the density of the phage is more than or equal to 150pfu/mL, the days with the highest temperature of 25-35 ℃ in 4-10 days in the future is more than or equal to 70%, the number of rainfall days is more than or equal to 40%, and the rice bacterial leaf streak of the field is predicted to occur in 4-10 days in the future; when the density of the phage is more than or equal to 150pfu/mL, but the highest temperature of the future weather forecast day is less than 24 ℃, the highest temperature is more than 35 ℃, the cumulative amount of cloudy clear days exceeds 50 percent, and the number of rainfall days is less than 40 percent, the occurrence of the rice bacterial leaf streak is delayed or prevented. The delay according to the present invention means that the disease is not temporarily developed within 7 to 14 days in the future as predicted.
The future weather of the invention can be known according to the weather forecast.
According to the continuous detection result, the occurrence period of the bacterial leaf streak of the rice can be accurately predicted by combining the growth period of the rice and the weather forecast of the medium and short periods.
The sample obtained in the step (1) is preferably immediately placed into a low-temperature sample box for preservation, so that the accuracy difference caused by the change of the quantity of the phage is avoided, the obtained sterile sample formed by the field water or the field mud is preferably reserved at 4 ℃, and the preservation time is not more than 48 h.
The sample obtained in step (1) of the present invention may be filter sterilized by a conventional method, for example, by using a 0.22 μm bacterial filter; adding 2-3 times of sterile water, shaking, standing for 10-15min, collecting supernatant, and filtering with 0.22 μm bacterial filter to remove bacteria.
Further, the sample collection method in the step (1) is preferably: selecting a rice planting field, randomly selecting 5 points in the field from the time when the rice is transplanted to turn green or the time when the direct seeding rice begins to tillere, sampling 20-100mL of field water at each point, and sampling 1 time at intervals of 5-7 days; and (3) entering the booting heading stage of the rice, randomly selecting 5 points in the field, sampling 20-100mL of field water at each point, and sampling 1 time at intervals of 3-5 days. Furthermore, a sterile container is selected for sampling, and sampling can be respectively added once before and after a typhoon storm. The obtained sample is quickly transferred into a low-temperature (2-8 ℃) sample box.
In some embodiments, the invention provides a method for preparing an indicator bacterium solution of the specific step (2), which comprises the following steps: inoculating a glycerol preservation indicator strain XZ45 at the temperature of-80 ℃ to an NA solid culture medium plate, and culturing for 36-48h at the temperature of 26-30 ℃; selecting a single colony to be inoculated into an NB liquid culture medium, culturing for 36-48h under the condition of 28-30 ℃, and oscillating at the frequency of 120-200 r/min; 1% (v/v) transfer of the culture broth to fresh NB brothCulturing at 28-30 deg.C for 12-30h, shaking at 120-200 r/min, and adjusting thallus concentration to 3.0 × 10 with sterile water8-2.0×109cfu/mL as an indicator bacterial liquid, in a more preferred preparation method, comprises (a) selecting a rice bacterial alternaria alternate strain XZ45 with stable genetic characteristics, strong pathogenicity and wide affinity as an indicator bacterial strain; (b) streaking and inoculating an indicator strain preserved by glycerol at the temperature of-80 ℃ to an NA solid culture medium plate, and culturing for 36-48h at the temperature of 28-30 ℃; (c) selecting a single colony to be inoculated into an NB liquid culture medium, and culturing for 36-48h under the conditions of 28-30 ℃ and 120-200 r/min; (d) transferring the culture solution into a fresh NB liquid culture medium at a rate of 1% (v/v), and culturing at 28-30 deg.C and 120-200 r/min for 18-24 h; (e) the cell concentration was adjusted to 3.0X 10 with sterile water8-2.0×109cfu/mL as indicator bacteria solution.
The indicator strain is preferably rice bacterial leaf streak germ XZ45, and the strain has wide phage affinity and stable genetic characteristics; the cell density is preferably 4.0X 108-1.2×109cfu/mL, more preferably 5.0X 108-1.2×109cfu/mL。
Further, the prepared indicator bacterium solution is used within 2 hours, and is more preferably used as it is.
In some embodiments, a solid media plate assay method is provided, comprising: (a) the sterile sample filtrate processed in the step (2) and the density of the bacterial cells are 4.0 multiplied by 108-1.2×109Adding cfu/mL of indicating bacterium liquid into a sterile culture dish, and uniformly mixing; (b) then adding 10-12mL of liquid NA culture medium at 40-45 ℃, rapidly mixing uniformly, performing inverted culture at 28-30 ℃ for 10-16h after solidification, investigating the number of plaques, and counting the density of phage in the field water or the field mud.
According to the continuous detection result, when the number of the phages in the paddy field water reaches or exceeds 150pfu/mL, the phages are shown to enter a sudden increase stage, and the occurrence period of the diseases can be accurately predicted by combining the future medium-short term weather forecast condition, the paddy growth period and the like.
Further, the mixing of the sterile sample obtained in the step (1) and the indicator bacterium liquid obtained in the step (3) is preferably completed within 48 hours, and more preferably within 36 hours.
In some embodiments of the invention, the NB media components are: 5g/L of peptone, 10g/L of sucrose, 1g/L of yeast extract, 3g/L of beef extract and 6.8-7.2 of pH; the preparation method of the NB culture medium comprises the steps of adding water into each component, fixing the volume to 1000mL, adjusting the pH value to 6.8-7.2, and sterilizing at 121 ℃ for 20 min. And adding 15g/L agar powder into the NB culture medium to obtain the NA solid culture medium.
The rice is indica rice variety, including conventional indica rice and hybrid indica rice.
The invention discloses the following technical effects:
the rice bacterial leaf streak bacterial strain XZ45 with stable biological characteristics and wide affinity is taken as an indicator bacterium, the problem that the affinity of the phage and the rice bacterial leaf streak bacterial strains from different sources has differentiation is solved, and the technical method for quickly and simply predicting the occurrence period of the rice bacterial leaf streak by using the phage is established. The rapid growth period of the phage is specified, and the growth period of the rice and the weather forecast of the medium and short periods are combined, so that the occurrence period of the rice bacterial leaf streak can be accurately predicted. Thereby improving the prevention and control effect of diseases, reducing the dosage of chemical pesticides and having better economic, ecological and social benefits.
Drawings
FIG. 1 shows the bacterial colony and bacterial cell morphology of rice bacterial streak germ XZ45 on NA medium plate;
FIG. 2 is a BLASTN search based on the 16S rRNA gene sequence of bacterial leaf streak germ XZ 45;
FIG. 3 is a plate test of bacterial rice streak germ XZ45 as an indicator strain for the number of phages in the field water;
FIG. 4 shows the detection of phage numbers in different regions of diseased field waters using indicator strain XZ 45.
Detailed Description
The following examples are provided to illustrate the present invention, but are not intended to limit the present invention. Unless otherwise specified, all technical methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples are all conventional reagent materials.
The invention relates to 7 strains of bacteriophage indicating strains rice bacterial streak germ (Xanthomonas oryzae pv. oryzicola, Xoc) Rs105, Xoc-192, Xoc-197, XZ45, Xoc-S, Xoc-M and Xoc-W are long-term storage strains in the research room, and promise to be permanently opened to the public, wherein the XZ45 strain has strong pathogenicity, stable biological characteristics, wide phage affinity and high sensitivity, is stored in the common microorganism center of China Committee for culture Collection of microorganisms at 3/8/2021, and has the strain preservation number of CGMCC No. 21877.
The NB media components described below were: 5g of peptone, 10g of sucrose, 1g of yeast extract and 3g of beef extract, adding water to a constant volume of 1000mL, adjusting the pH value to 6.8-7.2, and sterilizing at 121 ℃ for 20 min; 15g/L of agar powder is added into the NB culture medium, and the NA solid culture medium is obtained.
The following examples are only some of the preferred examples of the present invention, and are only for describing the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Example 1: identification of indicator Strain XZ45
(1) Morphological observation of indicator strain XZ 45: selecting thallus from XZ45 plate preserved at 4 deg.C by using sterile inoculating loop, streaking on NA solid culture medium plate, and culturing at 28 deg.C for 36-48h to obtain colony characteristics: the bacterial colony is smooth, opaque, glossy, round, convex and complete in edge; initially white and then light yellow to a diameter of 1-1.5 mm. Selecting thalli, observing the thalli by using a transmission electron microscope, wherein the morphological characteristics of the thalli are as follows: the thallus is short rod-shaped, and the size is (0.4-0.6) mu m multiplied by (1.1-2.0) mu m; no spore and capsule, and extracellular polysaccharide with mucilage outside the thalli; single generation, few pairs and no chain shape; one polar flagellum. The colony and cell morphology of XZ45 are shown in FIG. 1.
(2) Molecular characterization of indicator strain XZ 45: a single colony on an XZ45 culture plate is picked by using a sterilized toothpick and cultured in a 250mL triangular flask containing 50mL NB liquid medium for 36h at the temperature of 28 ℃ at 180r/min, and the total genomic DNA of XZ45 is extracted by using a bacterial genomic DNA extraction kit. The PCR product was sequenced by PCR amplification of DNA fragments using 16SF (5'-AGAGTTTGATCATGGCTCAG-3') and 16SR (5'-ACGGTTACCTTGTTACGACTT-3') universal primers for the bacterial 16S rRNA gene. Based on the comparison of BLSATN search in NCBI GenBank database based on the obtained 16S rRNA gene sequence (SEQ ID NO.1), it was found that the homology of XZ45 with the corresponding gene of bacterial leaf streak disease of Oryza sativa in NCBI GenBank database was as high as 99.8% or more (see FIG. 2).
Based on the colony, thallus morphology and molecular identification results, XZ45 can be determined to belong to rice bacterial streak pathogen (Xanthomonas oryzae pv. oryzicola). The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3, 8 and 2021, and the preservation number of the strain is CGMCC No. 21877.
Example 2: preparation of phage indicator bacterium liquid
Streaking indicator strains Rs105, Xoc-192, Xoc-197, XZ45, Xoc-S, Xoc-M, Xoc-W preserved by glycerol at the temperature of-80 ℃ on an NA solid culture medium plate, and culturing for 36-48h at the temperature of 28-30 ℃; selecting a single colony, inoculating the single colony into an NB liquid culture medium, and culturing for 36h at 28 ℃ under the condition of 180 r/min; transferring the culture solution into a fresh NB liquid culture medium at 1% (v/v), and culturing at 28 deg.C and 180r/min for 18-24 h; the cell concentration was adjusted to 5.0X 10 with sterile water8-1.2×109cfu/mL as indicator bacteria solution.
Example 3: screening of broad affinity indicator strains
The laboratory separates and preserves 17 strains of rice bacterial streak germ phage glycerol from different areas of Jiangsu, the strain number is shown in table 1 (the applicant promises to open and preserve samples to the public permanently), the phage strain which preserves the glycerol at the former stage at-80 ℃ absorbs 10 mu L respectively and adds the 10mL Rs105 bacterial liquid which is freshly cultivated, and the bacterial liquid is cultivated for 24-36h under the conditions of 28 ℃ and 180 r/min; centrifuging at 12000r/min at room temperature for 3min, collecting supernatant, filtering with 0.22 μm bacteria filter for sterilization, and diluting with sterile waterThe application is as follows. Adding 1.0mL of each phage liquid diluted in a gradient manner and 1.0mL of each indication bacterial liquid of example 2 into a disposable sterile culture dish respectively, and uniformly mixing; then adding 10-12mL of liquid NA culture medium cooled to 40-45 ℃, quickly mixing uniformly, performing inverted culture at 28 ℃ for 10-16h after solidification, investigating the number of plaques, and performing statistical analysis on the sensitivity of each indicator bacterium to phages from different sources. The results are shown in Table 1, the comprehensive detection sensitivity of the indicator strain XZ45 to 17 phage strains is highest, and the measured phage titer is (4.1X 10)10-2.7×1011) The plaque is transparent and clear when being cultured for 14h between pfu/mL, and the average diameter of the plaque formed by each plaque strain is 2.9mm-3.8 mm; the phage titer determined for the other 6 indicator strains was (1.4X 10)8-9.3×1010) Between pfu/mL, the resulting plaque diameter is between 1.7mm and 3.2 mm. These results show that the XZ45 indicator bacterium has wide affinity and high sensitivity to different phages, and formed plaques are clear and transparent and are convenient for counting statistics. In the following examples, XZ45 was used as an indicator strain for detecting bacteriophages of rice bacterial streak pathogens.
TABLE 1 detection sensitivity of different indicator strains to bacteriophages of rice bacterial streak blotch
Example 4: detection of phage density in different regional diseased field waters using indicator strain XZ45
In the heading stage of rice, field water is collected and disease indexes are investigated in 21 fields which generate bacterial leaf streak of rice in the persistent city of Jiangsu province and Huaian city respectively, 5 points are randomly selected for investigation and sampling in each field, 20mL of water sample is taken in each point by using a 150mL sterile plastic bottle, the total amount is 100mL, and the numbers of S1, S2, S3, S4, S5, S6, S7, S8, S9, H1, H2, 3, H4, H5, H6, H7, H8, H9, H10, H11 and H12 are respectively placed in a low-temperature (2-8 ℃) storage box and taken back to a laboratory. Meanwhile, the disease index of the bacterial leaf streak of the rice at 5 points of the sampled field is investigated, and the disease grading and investigating method comprises the following steps:
the sampling field was sampled at 5 points on a diagonal line, and 20 clumps of samples were investigated at each point, for a total of 100 clumps.
The classification criteria of diseased leaves are as follows:
level 0: the leaves have no disease spots;
level 1: the leaves only have small semitransparent water stain-like disease spots, which account for less than 1% of the area of the leaves;
and 3, level: the leaves have sporadic short and narrow scabs which account for 1 to 5 percent of the area of the leaves;
and 5, stage: the leaf has more scabs and occupies 6 to 25 percent of the leaf area;
and 7, stage: the scabs on the leaves are dense, and account for 26-50% of the leaf area;
and 9, stage: the scabs of the leaves are densely distributed, the area of the leaves is more than 51 percent, and the leaves become orange brown, curled and withered.
The disease index calculation method comprises the following steps:
collected field water was subjected to filtration sterilization using a 0.22 μm bacterial filter, and was diluted 5-fold in sterile water. The bacterial leaf streak disease of rice was activated, XZ45, and a phage indicator solution was prepared according to the method described in "example 2" above. Respectively adding 1mL of sample gradient diluent and 1mL of fresh indicator bacterium liquid into a sterile culture dish, and uniformly mixing; then adding 10-12mL of liquid NA culture medium at 40-45 ℃, rapidly mixing uniformly, performing inverted culture at 28 ℃ for 12-16h after solidification, investigating the number of plaques, and counting the density of phage in field water. As a result, the disease indexes of 21 disease fields are 0.8-35.8, phages are detected in the field water of all the fields, the phage density reaches 870-129000pfu/mL, and the quantity of the phages is basically and positively correlated with the disease indexes of the investigation fields, as shown in figures 3 and 4. The above results again show that the indicator strain XZ45 has broad phage affinity and that the plaques formed are large and easy to investigate.
Example 5: method for predicting emergence period of rice bacterial leaf streak in field by using bacteriophage
3 hybrid indica rice planting field blocks with the history of the bacterial leaf streak of rice are selected from Xuyi county Ma dam town, Mu shop town and Siyang county Zhengchang in Jiangsu province, wherein the species are respectively C Liangyou 998, Y Liangyou 1998, Huiyou 898, and are transplanted in 10-15 days in 6 months. The occurrence of bacterial leaf streak in rice and the density of phages in field water were investigated from 25/6, and the investigation and sampling were carried out according to the method described above in "example 4" and 1 sampling was carried out at 5-7 days intervals. The obtained sample is quickly transferred into a low-temperature (2-8 ℃) sample box. Collected field water samples were filter sterilized using a 0.22 μm bacterial filter, and the sterile filtrate was diluted 5-fold in gradient with sterile water. The bacterial leaf streak disease of rice was activated, XZ45, and a phage indicator solution was prepared according to the method described in "example 2" above. Respectively adding 1mL of sample gradient diluent and 1mL of fresh indicator bacterium liquid into a sterile culture dish, and uniformly mixing; then adding 10-12mL of liquid NA culture medium at 40-45 ℃, rapidly mixing uniformly, and performing inverted culture for 12-16h at 28 ℃ after solidification; and (5) investigating the number of the plaques, and counting the density of the phage in the field water. During the investigation period, the rice is in the tillering stage; 14 days after 7 months and 9 days, the days with the highest daily temperature of 25-35 ℃ are 13-14 days, and the days of rainfall are 7-12 days. The investigation on 7 months and 9 days shows that the density of the phages in the three fields respectively reaches 184pfu/mL, 226pfu/mL and 279pfu/mL, the phages enter a sudden increase period and are attacked on the 10 th day, the 7 th day and the 12 th day after the densities reach the densities respectively, and the phages meet the prediction result of the occurrence of the bacterial streak of the paddy rice 7 to 14 days after the phages enter the sudden increase period. See table 2 specifically:
TABLE 2 dynamic behavior of bacteriophages in paddy water and the occurrence period of bacterial leaf streak in paddy rice
Example 6: method for predicting emergence period of rice bacterial leaf streak in field by using bacteriophage
In Xuyi county Ma Zhen, mu shop Zhen, guan Tan Zhen Si Yang county Zhen. The density of phages in the field was investigated from 6/8 to predict the occurrence period of rice bacterial leaf streak, and the initial occurrence time of rice bacterial leaf streak was also investigated. Investigation and sampling were carried out as described above in "example 4", with 1 sampling at 5-7 day intervals, and the samples taken were quickly transferred to a low temperature (2-8 ℃) sample box. Collected field water samples were sterilized by filtration using a 0.22 μm bacterial filter and diluted 5-fold in sterile water. The bacterial leaf streak disease of rice was activated, XZ45, and a phage indicator solution was prepared according to the method described in "example 2" above. Respectively adding 1mL of sample gradient diluent and 1mL of fresh indicator bacterium liquid into a sterile culture dish (with the diameter of 9cm), and uniformly mixing; then adding 10-12mL of liquid NA culture medium at 40-45 ℃, rapidly mixing uniformly, performing inverted culture at 28 ℃ for 12-16h after solidification, investigating the number of plaques, and counting the density of phage in field water. During the investigation period, the rice is in the stage from the booting stage to the heading and flowering stage. During the period from 6 days at 8 months to 20 days at 8 months, the number of high-temperature or cloudy sunny days at the temperature of more than 35 ℃ is accumulated for 8 to 11 days, and the number of rainfall days is 3 to 5 days; 8 months and 20 days to 9 months and 3 days, wherein the days with the highest temperature of 25-35 ℃ are 13-14 days, the rainfall days are 7-9 days, and the dew is heavy. As a result, it was found that the densities of phages in plots No.1, No.2, No. 3, No. 5 and No. 7 reached 167pfu/mL, 203pfu/mL, 350pfu/mL, 281pfu/mL and 413pfu/mL, respectively, and then the plots entered the sharp increase phase, and the rice bacterial leaf streak occurred in succession in each of the plots from day 4 to day 8 after the sharp increase phase. On 6 days 8 months, the density of phage in No. 6 field water of the cultured C Liangyou 0861 is 277pfu/mL, which reaches the standard of phage sharp increase period, but the rice bacterial leaf streak occurs on the 16 th day because the disease occurrence is delayed by high-temperature and rainy days before 20 days 8 months. During the investigation period, the highest phage density of the No. 4 and No. 8 fields is 67pfu/mL and 121pfu/mL respectively, and the standard of the phage sharp increase stage is not reached; by the late stage of maturity, no bacterial leaf streak of rice occurred in both fields. In the heading and flowering period of the rice, if the weather meets the condition of the invention, the bacterial leaf streak of the rice occurs, and the number of the phage reaches 150pfu/mL, the disease occurs within 4-10 days after the sudden increase, and the disease occurs in a delayed way due to high temperature and drought. The field investigation results are consistent with the results of the invention. See in particular table 3 below:
TABLE 3 dynamic behavior of bacteriophages in paddy fields and the occurrence period of bacterial leaf streak in paddy fields
Sequence listing
<110> agricultural science and academy of Jiangsu province
<120> an indicator strain and a method for predicting the emergence period of rice bacterial leaf streak by using bacteriophage
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1510
<212> DNA
<213> Xanthomonas oryzae Dastarchya pathogenic variants (Xanthomonas oryzae pv. oryzicola)
<400> 1
acggttacct tgttacgact tcaccccagt catcggccac accgtggcaa gcgccctccc 60
gaaggttaag ctacctgctt ctggtgcaac aaactcccat ggtgtgacgg gcggtgtgta 120
caaggcccgg gaacgtattc accgcagcaa tgctgatctg cgattactag cgattccgac 180
ttcatggagt cgagttgcag actccaatcc ggactgagat agggtttctg ggattggctt 240
gccctcgcgg gtttgcagcc ctctgtcccc accattgtag tacgtgtgta gccctggtcg 300
taagggccat gatgacttga cgtcatcccc accttcctcc ggtttgtcac cggcggtctc 360
cttagagttc ccaccattac gtgctggcaa ctaaggacaa gggttgcgct cgttgcggga 420
cttaacccaa catctcacga cacgagctga cgacagccat gcagcacctg tctcacggtt 480
cccgaaggca ccaatccatc tctggaaagt tccgtggatg tcaagaccag gtaaggttct 540
tcgcgttgca tcgaattaaa ccacatactc caccgcttgt gcgggccccc gtcaattcct 600
ttgagtttca gtcttgcgac cgtactcccc aggcggcgaa cttaacgcgt tagcttcgat 660
actgcgtgcc aaattgcacc caacatccag ttcgcatcgt ttagggcgtg gactaccagg 720
gtatctaatc ctgtttgctc cccacgcttt cgtgcctcag tgtcagtgtt ggtccaggta 780
gccgccttcg ccacggatgt tcctcccgat ctctacgcat ttcactgcta caccgggaat 840
tccgctaccc tctaccacac tctagtgacc cagtatccac tgcaattccc aggttgagcc 900
cagggctttc acaacagact taaaccacca cctacgcacg ctttacgccc agtaattccg 960
agtaacgctt gcacccttcg tattaccgcg gctgctggca cgaagttagc cggtgcttat 1020
tctttgggta ccgtcagaac aatcgggtat taaccgactg cttttctttc ccaacaaaag 1080
ggctttacaa cccgaaggcc ttcttcaccc acgcggcatg gctggatcag gcttgcgccc 1140
attgtccaat attccccact gctgcctccc gtaggagtct ggaccgtgtc tcagttccag 1200
tgtggctgat catcctctca gaccagctac ggatcgtcgc cttggtgggc ctttaccccg 1260
ccaactagct aatccgacat cggctcattc aatcgcgcga agcccgaagg tcctccgctt 1320
tcacccgtag gtcgtatgcg gtattagcgt aagtttccct acgttatccc ccacgaaaga 1380
gtagattccg atgtattcct cacccgtccg ccactcgcca cccataagag caagctctta 1440
ctgtgctgcc gttcgacttg catgtgttag gcctgccgcc agcgttcact ctgagccatg 1500
atcaaactct 1510
Claims (10)
1. A bacterial rice streak bacterial strain XZ45 is classified as Xanthomonas oryzae pathogenic variant (Xanthomonas oryzae pv. oryzicola) with the preservation number of CGMCC No. 21877.
2. Use of the bacterial leaf streak strain XZ45 according to claim 1 for predicting the stage of occurrence of bacterial leaf streak in rice.
3. A method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage, which is characterized by comprising the following steps:
(1) sample collection and preparation: collecting field water for 1 time every 3-10 days from the time of transplanting and turning green or the tillering stage of direct-seeding rice, and filtering and sterilizing to form a sterile sample; if the field is anhydrous, collecting field mud for replacement, adding sterile water with the weight 2-3 times of that of the field mud, uniformly mixing, standing, taking supernatant, and filtering for sterilization to form a sterile sample;
(2) preparing an indication bacterial liquid: inoculating the strain XZ45 of claim 1 into NB liquid medium, culturing at 28-30 deg.C for 36-48h, shaking at 120-200 r/min, and adjusting thallus concentration to 3.0 x 10 with sterile water8-2.0×109cfu/mL as an indicator bacterium liquid;
(3) and (3) phage detection: adding 0.1-2.0mL of the sterile sample in the step (1) and 0.5-2mL of the indication bacterial liquid in the step (2) into a sterile culture dish, uniformly mixing, then adding a liquid NA culture medium at 40-45 ℃, rapidly and uniformly mixing, performing inverted culture for 10-16h at 28-30 ℃ after solidification, investigating the number of plaques, and counting the density of phage in field water or mud;
(4) prediction of the emergence period of rice bacterial leaf streak: when the rice is in a tillering stage, when the density of phage is more than or equal to 150pfu/mL, the day with the highest temperature of 25-35 ℃ in 7-14 days in the future is more than or equal to 70%, the number of rainfall days is more than or equal to 40%, and the rice bacterial leaf streak of the field is predicted to occur in 7-14 days in the future; when the rice is in the booting stage, when the density of the phage is more than or equal to 150pfu/mL, the days with the highest temperature of 25-35 ℃ in 4-10 days in the future is more than or equal to 70%, the number of rainfall days is more than or equal to 40%, and the rice bacterial leaf streak of the field is predicted to occur in 4-10 days in the future; when the density of the phage is more than or equal to 150pfu/mL, but the highest temperature of the future weather forecast day is less than 24 ℃, the highest temperature is more than 35 ℃, the cumulative amount of cloudy clear days exceeds 50 percent, and the number of rainfall days is less than 40 percent, the occurrence of the rice bacterial leaf streak is delayed or prevented.
4. The method for predicting the occurrence period of bacterial leaf streak of rice using bacteriophage according to claim 3, wherein the sample obtained in step (1) is immediately stored in a low temperature sample box, preferably a sterile sample of the obtained field water or mud is kept at 4 ℃ for a period of time not exceeding 48 hours.
5. The method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage according to claim 3, wherein the sample collection method of step (1) is: selecting a rice planting field, randomly selecting 5 points in the field from the time when the rice is transplanted to turn green or the time when the direct seeding rice begins to tillere, sampling 20-100mL of field water at each point, and sampling 1 time at intervals of 5-7 days; and (3) entering the booting heading stage of the rice, randomly selecting 5 points in the field, sampling 20-100mL of field water at each point, and sampling 1 time at intervals of 3-5 days.
6. The method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage according to claim 3, wherein the sample collection in step (1) is performed by sampling in a sterile container, and the sampling is performed once before and after typhoon rainstorm; preferably, the sample taken is quickly transferred to a low temperature (2 ℃ to 8 ℃) sample box.
7. The method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage according to claim 3, wherein the preparation method of the indicator bacterial liquid of step (2) comprises: inoculating a glycerol preservation indicator strain XZ45 at the temperature of-80 ℃ to an NA solid culture medium plate, and culturing for 36-48h at the temperature of 26-30 ℃; selecting a single colony to be inoculated into an NB liquid culture medium, culturing for 36-48h under the condition of 28-30 ℃, and oscillating at the frequency of 120-200 r/min; transferring the culture solution into fresh NB liquid culture medium at 1% (v/v), culturing at 28-30 deg.C for 12-30h, shaking at 120-200 r/min, and adjusting thallus concentration to 3.0 × 10 with sterile water8-2.0×109cfu/mL as indicator bacteria solution.
8. The method according to claim 7, wherein the method comprises selecting a plant having stable genetic characteristics,The rice bacterial leaf streak strain XZ45 with strong pathogenicity and wide compatibility is taken as an indicator strain; (b) streaking and inoculating an indicator strain preserved by glycerol at the temperature of-80 ℃ to an NA solid culture medium plate, and culturing for 36-48h at the temperature of 28-30 ℃; (c) selecting a single colony to be inoculated into an NB liquid culture medium, and culturing for 36-48h under the conditions of 28-30 ℃ and 120-200 r/min; (d) transferring the culture solution into a fresh NB liquid culture medium at a rate of 1% (v/v), and culturing at 28-30 deg.C and 120-200 r/min for 18-24 h; (e) the cell concentration was adjusted to 3.0X 10 with sterile water8-2.0×109cfu/mL as indicator bacteria solution.
9. The method for predicting the stage of occurrence of bacterial leaf streak of rice using bacteriophage according to claim 3, wherein the concentration of bacterial cells is adjusted to 4.0X 10 using sterile water in the step (2)8-1.2×109cfu/mL, preferably 5.0X 108-1.2×109cfu/mL; the prepared indicator bacterium solution is preferably used within 2 hours, and more preferably is ready to use.
10. The method for predicting the occurrence period of rice bacterial leaf streak by using bacteriophage according to claim 3, wherein the step (1) of collecting the sample from the step (3) of sterile sample and the indicator bacteria liquid are mixed within 48h, preferably within 36 h.
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