A rapid identification method for rubber tree powdery mildew conidia

Abstract

The invention provides a method for quickly identifying conidia of rubber tree powdery mildew, comprising: (1) collecting conidia; (2) preparing conidia into a spore suspension, measuring the spontaneous fluorescence intensity of the conidia under red, green, blue and cyan channels respectively, expressing it in grayscale values, and screening strains with spontaneous fluorescence intensity characteristics; (3) taking the strains screened out in step 2 as objects, scanning the fluorescence spectrum within the wavelength of 300-430nm when the excitation light Em=455nm and the wavelength of 400-700nm when the emission light Ex=375nm, and judging the rubber tree powdery mildew according to the fluorescence intensity of the spontaneous fluorescence peak value. The invention combines microscopic observation of different fluorescence channels of the rubber tree powdery mildew with the fluorescence spectrum, realizes rapid and accurate identification and appraisal of the rubber tree powdery mildew, and provides strong support for the prediction and forecast of rubber tree powdery mildew and disease prevention and control.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a method for quickly identifying conidia of powdery mildew on rubber trees. Background Art

[0002] Natural rubber is an important strategic material in my country and an important economic crop in tropical areas of my country. The leaves of rubber trees have long been infected by powdery mildew, which is one of the most serious pathogens causing leaf diseases. Rubber tree powdery mildew is a typical airborne disease. Its strong infection ability and wide range of spread make rubber tree powdery mildew endanger various rubber tree plantations in my country, affect natural rubber production, and bring serious economic losses to crops. Not only that, the pathogen also has the biological characteristics that cannot be completely cultured in vitro, and is a dedicated parasitic fungus, which makes the purification and cultivation of rubber tree powdery mildew face serious obstacles.

[0003] For a long time, traditional monitoring and morphological identification have mainly relied on spore catchers and conidia morphology recognition, which is not only time-consuming and labor-intensive, but also limited by space and disease time. In addition, powdery mildew conidia have extremely similar morphological characteristics, making it difficult to identify them at the species level. Even with the use of molecular biological identification techniques such as single gene sequence analysis and multi-gene tandem evolution analysis, there are still problems such as ineffectiveness against single spores and difficulty in pure culture of the obligate parasitic fungus powdery mildew and insufficient sample quantity. The existing traditional rubber tree powdery mildew research and control methods provide limited references and are difficult to meet strategic needs. It is urgent to develop high-throughput monitoring and early warning technologies to provide leading prevention and control.

[0004] Specific optical characteristics and physiological and biochemical indicators are the basis of intelligent monitoring. In organisms, the types and numbers of endogenous fluorophores are limited. The physiological and biochemical conditions and positioning information that change over time can be associated and tracked through the autofluorescence of endogenous fluorophores, and the biochemical properties and metabolic activities can be mapped in space and time, which is of great significance to the study of fungal cell biology and metabolism. Endogenous fluorophores are closely related to energy metabolism, pathogenic mechanisms, etc. At present, endogenous fluorophores identified in fungi include flavin, NAD(P)H and melanin. However, there are few studies on the use of fungal spore autofluorescence to achieve differentiation and identification, and the research level is still shallow. There is still a blank in the study of conidia autofluorescence of the obligate parasite powdery mildew. Rubber tree powdery mildew is an important obligate parasitic plant pathogenic fungus. It relies on airborne infection to infect rubber tree leaves, causing a large reduction in natural rubber production every year. Its rapid and accurate identification can provide strong support for the prevention and control of rubber tree disasters. Summary of the invention

[0005] In view of this, the present invention proposes a method for rapid identification of rubber tree powdery mildew conidia, which utilizes microscopic observation of different fluorescence channels of rubber tree powdery mildew in combination with fluorescence spectroscopy to achieve rapid and accurate identification and authentication of rubber tree powdery mildew, thereby making up for the deficiencies in existing methods and technologies for identifying and authenticating rubber tree powdery mildew, and providing a new idea and method for identifying rubber tree powdery mildew.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a method for quickly identifying conidia of powdery mildew on rubber trees, comprising the following steps:

[0008] S1. Collecting conidia of pathogenic fungi and / or powdery mildew of rubber trees;

[0009] S2. Measurement of fluorescence intensity of conidia: The collected conidia were made into a spore suspension, and the spontaneous fluorescence intensity of conidia was measured under the red, green, blue and cyan channels with an exposure time of 100 ms using a fluorescence microscope equipped with DP80CCD and U-HGLGPS light source, respectively. The intensity was expressed as grayscale value, and strains with spontaneous fluorescence intensity characteristics were screened out;

[0010] S3, fluorescence spectrum determination of conidia: taking the strain screened out in step 2 as the object, taking 0.1-0.3g of its solid conidia, respectively, under the wavelength of 300-430nm of the excitation light Em=455nm, and the wavelength of 400-700nm of the emission light Ex=375nm, the rubber tree powdery mildew was judged according to the fluorescence intensity of the autofluorescence peak.

[0011] Further, in step S1, the collection of conidia includes collection and / or culture methods.

[0012] Further, in step S2, the concentration of the spore suspension is 10 5 CFU / mL.

[0013] To further illustrate, in step S2, the intensity of the autofluorescence is expressed as a gray value using ImageJ software, and the gray value of the strain with the autofluorescence intensity characteristic is greater than 10.

[0014] Further, in step S3, the speed of the fluorescence spectrum scanning is 1200 nm / min, the slit width is 10 nm, and the photomultiplier tube voltage is 400 V PMT.

[0015] It is further explained that in step S3, the peak position and peak value of the spontaneous fluorescence intensity of rubber tree powdery mildew in the excitation spectrum with a wavelength of 300-430nm are 373nm, 1813.2au, which is intensity specific compared with the excitation spectra of other types of powdery mildew.

[0016] It is further explained that in step S3, the peak position and peak value of the spontaneous fluorescence intensity of rubber tree powdery mildew in the emission spectrum with a wavelength of 400-700nm are 373nm, 1927.2au, which is specific in intensity compared with the emission spectra of other types of powdery mildew.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention is the first to utilize the spontaneous fluorescence intensity in combination with the fluorescence spectrum characteristics, and utilizes microscopic observation and spectral measurement, from microscopic conidia fluorescence intensity observation to quantitative solid spore spectral scanning, to distinguish and identify rubber tree powdery mildew, thereby effectively improving the accuracy and efficiency of identification.

[0019] The experimental results of the present invention show that the spontaneous fluorescence of rubber tree powdery mildew conidia has certain specificity in intensity, and produces a spontaneous fluorescence phenomenon that is significantly stronger than that of other powdery mildew conidia under different fluorescence channels. At the same time, through the spectral emission peak intensity and its spectral specificity, accurate and rapid identification of rubber tree powdery mildew is achieved.

[0020] In practical applications, the present invention can diversify the collection methods, and the collected samples do not need to be separated and purely cultured, and do not need to be pre-enriched. The sample source is not limited to laboratory conditions, and can be directly collected from the surface of diseased leaves in the wild or from the air. There is no need to rely on inducing the production of specific fungal structures. This effectively solves the problems faced by traditional morphological identification methods at the gene level, such as the similarity of conidia morphology, the difficulty in in vitro pure culture due to obligate parasitism, and the difficulty in collecting samples, thereby making up for the shortcomings of traditional morphological identification.

[0021] The identification method of the invention is intuitive, efficient and highly accurate, and requires a small amount of samples. Especially for rubber tree powdery mildew and other types of powdery mildew, which are difficult to culture and are obligate parasites, a small amount of conidia can meet the identification needs.

[0022] The present invention achieves the expected effect by constructing a rubber tree powdery mildew conidia identification technology and selecting the best parameter settings, thereby providing strong support for rubber tree powdery mildew prediction and disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The autofluorescence microscopic observation images of conidia of different plant pathogenic fungi in the embodiments of the present invention under different fluorescence channels;

[0024] Figure 2 : A is a comparative analysis diagram of the autofluorescence intensity of conidia of different plant pathogenic fungi in the embodiments of the present invention; A is a comparison of the autofluorescence intensity of conidia of three kinds of powdery mildew; B is a comparison of the autofluorescence intensity of conidia of three kinds of rubber tree pathogenic fungi;

[0025] Figure 3 The fluorescence spectrum of the bacteria with autofluorescence characteristics according to the embodiment of the present invention, A is the excitation spectrum, and B is the emission spectrum;

[0026] Figure 4 The present invention is a flowchart of the rapid identification of rubber tree powdery mildew conidia according to an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0030] In practical applications, the culture method, collection method, materials used in the process, etc. of the powdery mildew conidia used in the present invention are not limited to specific methods and brands. For example, spores can be captured from the air, inoculated into a culture medium to produce spores, collected from the diseased site, etc., without the need for filtering, washing, and concentration adjustment.

[0031] Embodiment 1-collecting the conidia of pathogenic fungi and / or powdery mildew of rubber tree:

[0032] The rubber tree powdery mildew (Erysihe quercicola) was inoculated on the bronze-stage rubber leaves, cultured and collected, and other pathogenic fungi were inoculated on the surface of pad4 mutant Arabidopsis leaves and cowpea leaves, cultured and collected; or inoculated on PDA culture medium, cultured at 28°C for three days, then the hyphae were scraped off and placed under light conditions to induce spore production.

[0033] All conidia were suspended in sterile water and filtered to remove impurities such as hyphae. Then, they were centrifuged for 3 minutes, the supernatant was removed, and the mixture was washed three times. The concentration of the spore suspension was diluted to 1×10 5 CFU / mL, for future use.

[0034] Example 2 - Fluorescence intensity measurement of conidia

[0035] The concentration of Example 1 was 10 5The conidia suspension of 100 CFU / mL was dropped onto a glass slide, and an Olympus BX53 fluorescence microscope equipped with a DP80 CCD and U-HGLGPS light source was used to measure the spontaneous fluorescence intensity of the conidia under four channels, red (RFP), green (GFP), blue (DAPI) and cyan (CFP), with an exposure time of 100 ms. The fluorescence intensity was calculated using ImageJ and expressed as a grayscale value, and the strains with spontaneous fluorescence intensity characteristics (grayscale value greater than 10) were screened out.

[0036] Example 3 - Fluorescence spectroscopy characterization of conidia

[0037] According to the conidia fluorescence specificity detection results of Example 2, the strains screened in Example 2 were used as the object, 0.1 g of each of the three fresh solid strains cultured for 14-21 days were taken, and the collected spores were fixed using the solid powder clamp supporting the F-7000 fluorescence spectrometer.

[0038] Fluorescence spectrum scanning was performed, and the excitation spectrum (Em=455nm) and emission spectrum (Ex=375nm) of the spores were scanned in the range of 300-430nm and 400-700nm, respectively, with a scanning speed of 1200nm / min, a slit width of 10nm, and a photomultiplier tube voltage of 400V PMT. Each sample was measured 5 times and an average value was taken, and the rubber tree powdery mildew was judged according to the fluorescence intensity of the autofluorescence peak value. The peak value of the autofluorescence intensity of the rubber tree powdery mildew in the excitation spectrum with a wavelength of 300-430nm was 373nm, 1813.2au; the peak position and peak value of the autofluorescence intensity of the rubber tree powdery mildew in the emission spectrum with a wavelength of 400-700nm were 373nm, 1927.2au.

[0039] Embodiment 4-rubber tree powdery mildew conidia rapid identification method

[0040] (1) Collection of conidia of plant pathogenic fungi:

[0041] The experimental subjects were Erysihe quercicola, Erysihe cichoracearum, Podosphaera hibiscicola, Colletotrichum tropicalale and Lasiodiplodia theobromae.

[0042] The rubber tree powdery mildew (Erysihe quercicola) was inoculated on the bronze stage rubber leaves, cultured and collected. As powdery mildew controls, the powdery mildew Erysihe cichoracearum and Podosphaera hibiscicola were inoculated on the surface of pad4 mutant Arabidopsis leaves and cowpea leaves, cultured and collected.

[0043] Colletotrichum tropicale and Lasiodiplodia theobromae, both of which are diseases of the rubber tree leaves, were inoculated into PDA medium and cultured at 28°C for three days. After that, the mycelium was scraped off and placed under light conditions to induce spore production.

[0044] All the conidia collected above were suspended in sterile water and filtered through Miracloth filter cloth (Merck KgaA, pore size 22-25 μm, Germany) to remove impurities such as hyphae; then centrifuged at 10,000 rpm for 3 minutes to remove the supernatant, washed three times, and diluted the spore suspension to a concentration of 1×10 5 CFU / mL, for future use.

[0045] (2) Observation of fluorescence specificity of conidia of plant pathogenic fungi

[0046] Identification can be achieved by utilizing the specificity of the spontaneous fluorescence of rubber powdery mildew. Take the spore suspension collected and prepared in step (2), add 10-20 μL of the conidia suspension onto a glass slide, and use an Olympus BX53 fluorescence microscope equipped with a DP80CCD and a U-HGLGPS light source to observe and take pictures of the spontaneous fluorescence of the conidia for fluorescence intensity calculation under four channels: red (RFP), green (GFP), blue (DAPI), and cyan (CFP). The spontaneous fluorescence microscopic observation of the conidia of rubber powdery mildew (E.quercicola), E.cichoracearum, P.hibiscicola, C.tropicale, and L.theobromae under four channels is achieved, with an exposure time of 100 ms. ImageJ is used to represent the fluorescence intensity with grayscale values, and the statistical fluorescence intensity is calculated, as shown in FIG. Figure 1 shown.

[0047] The conidia autofluorescence intensity measurement of each fungus included three biological replicates, and each biological replicate contained the fluorescence intensity of 10 single spores and averaged them to screen out fungal species with autofluorescence characteristics.

[0048] The results are as follows Figure 1 , E.quercicola, C.siamense and L.theobromae are all pathogenic fungi that infect rubber trees and have the same host and physiological environment. However, the spores of C.siamense and L.theobromae did not show visible autofluorescence, while the conidia of E.quercicola showed significantly stronger autofluorescence.

[0049] The three powdery fungi, Erysihe quercicola, E. cichoracearum, and P. hibiscicola, all showed a certain degree of autofluorescence, indicating that autofluorescence may be a common characteristic of powdery fungi. In addition, although the morphology of the three powdery fungi is very similar, E. quercicola has a significantly stronger autofluorescence, which is a clear sign to distinguish it from the other two powdery fungi.

[0050] like Figure 2 As shown, in order to better reflect its intensity, the intensity of the autofluorescence is counted in the ImageJ software and expressed as grayscale value. Among them, the spore autofluorescence with a grayscale value lower than 10 cannot be observed by naked eyes, therefore, the strains with a grayscale value greater than 10 with autofluorescence characteristics are screened for the next step of fluorescence spectrum characterization (rubber powdery mildew (Erysihe quercicola), two spore powdery mildew (E. cichoracearum), fork thread single capsule powdery mildew (P. hibiscicola)).

[0051] In the figure, lowercase letters represent 95% difference level, uppercase letters represent 99% difference level, and data with the same letters have no significant difference.

[0052] (3) Fluorescence spectroscopy characterization of rubber tree powdery mildew

[0053] The fluorescence spectrum characteristics of rubber tree powdery mildew are used to achieve species-level identification. According to the spontaneous fluorescence microscopic observation results of step (2), rubber tree powdery mildew (Erysihe quercicola), two-spored powdery mildew (E. cichoracearum), and forked thread single capsule powdery mildew (P. hibiscicola) are used as experimental objects, and 0.1g of each of the three kinds of fresh powdery mildew collected and cultured for 14-21 days is collected, and the collected spores are fixed using a solid powder clip supporting an F-7000 fluorescence spectrometer.

[0054] Fluorescence spectra of three powdery mildew species were scanned. The excitation spectrum (Em=455nm) and emission spectrum (Ex=375nm) of the spores were scanned in the range of 300-430nm and 400-700nm, respectively, with a scanning speed of 1200nm / min, a slit width of 10nm, and a photomultiplier tube voltage of 400V PMT. Each sample was measured 5 times and the average value was taken.

[0055] The results are as follows Figure 3 As shown, the fluorescence spectra of the conidia of the three powdery mildews all showed obvious peaks. In their excitation spectra, they had similar maximum excitation wavelengths in the range of 360-375nm; in their emission spectra, they had extremely similar maximum emission wavelengths in the range of 458-463nm. It is worth noting that the rubber tree powdery mildew has an obvious high-intensity autofluorescence peak, which shows that the autofluorescence of the rubber tree powdery mildew is specific and significantly stronger than the other two powdery mildews, which is consistent with the measurement results in step 2. In addition, the powdery mildew of the fork thread monocysts showed a clear trend of different emission spectra at 487nm, and it was not only different from the other two powdery mildews in intensity. The peak position and peak value of the spontaneous fluorescence intensity of the rubber tree powdery mildew Erysihequercicola in the excitation spectrum of the wavelength of 300-430nm are 373nm, 1813.2au; the peak position and peak value of the spontaneous fluorescence intensity of the rubber tree powdery mildew in the emission spectrum of the wavelength of 400-700nm are 373nm, 1927.2au.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A rapid identification method for rubber tree powdery mildew conidia, characterized in that: The steps include: S1. Collect conidia of pathogenic fungi of rubber trees; S2. Measurement of fluorescence intensity of conidia: The collected conidia were made into a spore suspension, and the spontaneous fluorescence intensity of conidia was measured under the red, green, blue and cyan channels using a fluorescence microscope equipped with a DP80 CCD and a U-HGLGPS light source, respectively, and expressed as grayscale values ​​to screen out bacterial species with spontaneous fluorescence intensity characteristics; S3, fluorescence spectrum determination of conidia: taking the strain screened out in step 2 as the object, taking 0.1-0.3g of solid conidia, respectively, in the wavelength of the excitation light Em=455nm is 300-430nm, the wavelength of the emission light Ex=375nm is 400-700nm range, carry out fluorescence spectrum scanning, judge rubber tree powdery mildew according to the fluorescence intensity of the autofluorescence peak value; In step S2, the concentration of the spore suspension is 10 5 CFU / mL; The gray value of the strains screened for autofluorescence intensity characteristics was greater than 10; In step S3, the peak position and peak value of the spontaneous fluorescence intensity of the rubber tree powdery mildew in the excitation spectrum with a wavelength of 300-430nm are 373nm, 1813.2au; In step S3, the peak position and peak value of the spontaneous fluorescence intensity of the rubber tree powdery mildew in the emission spectrum with a wavelength of 400-700 nm are 373 nm and 1927.2 au.

2. a kind of rubber tree powdery mildew conidia rapid identification method as claimed in claim 1, it is characterized in that: In step S1, the collection of conidia includes collection and / or culture methods.

3. a kind of rubber tree powdery mildew conidia rapid identification method as claimed in claim 1, it is characterized in that: In step S2, the intensity of autofluorescence was expressed as grayscale value using ImageJ software, and the exposure time was 100 ms.

4. a kind of rubber tree powdery mildew conidia rapid identification method as claimed in claim 1, is characterized in that: In step S3, the fluorescence spectrum scanning speed is 1200 nm / min, the slit width is 10 nm, and the photomultiplier tube voltage is 400 VPMT.

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