Method for detecting pectinolytic pathogens of crops and ornamental plants, microbial medium for use in this method and test kit implementing this method

Abstract

The subject matter of the present invention is a method for detecting pectinolytic pathogens of crops and ornamental plants, including the composition of the microbial medium used in the method and a test kit that allows the use of the above-mentioned method, including under field conditions. The method is based on the hydrolysis of a fluorescently labelled substrate by a pectinolytic enzyme. The product can be separated by migration on a membrane.

Description

[0001] METHOD FOR DETECTING PECTINOLYTIC PATHOGENS OF CROPS AND ORNAMENTAL PLANTS, MICROBIAL MEDIUM FOR USE IN THIS METHOD AND TEST KIT IMPLEMENTING THIS METHOD

[0002] DESCRIPTION

[0003] The present invention relates to a method for detecting pectinolytic plant pathogens, including phytopathogens of the Soft Rot Pectobacteriaceae (SRP) group, in plant tissue and other matrices, which can be used under field conditions. The present invention also relates to a microbial medium used in the said method and to a test kit implementing the method.

[0004] STATE OF THE ART

[0005] The most common phytopathogens that are etiological agents of soft rot are bacteria currently classified in the genera Pectobacterium and Dickeya, which belong to the family Pectobacteriaceae (SRP). Plant diseases caused by bacteria from the SRP group are an economically significant problem. SRP infect crop plants and contribute to significant crop and stored yield losses. The problem of soft rot affects producers of agricultural products, buying brokers and sellers. Symptoms of infection and rapidly progressive maceration of plant tissue are often only visible during crop storage, so it is important to quickly detect the presence of the pathogen and eliminate infected plants. Initially, the detection of these bacteria was based mainly on the isolation of live bacterial cells on a solid, selective microbial medium, followed by further analyses such as microscopic examination, biochemical or serological tests. Over time, faster, more specific and reproducible molecular techniques based on detection of specific regions of the SRP genome have been introduced [1], Currently, there are a number of techniques allowing detection of these bacteria; the most commonly used are culture and genetic methods.

[0006] Classical culture methods allow the isolation and identification of SRP bacteria. For this purpose, CVP (Crystal Violet Pectate) medium [2], or its modifications, are used. A single- or double-layer medium is used. The selectivity of CVP is based on the use of pectin as the primary carbon source and the addition of crystal violet, which inhibits the growth of most Gram-positive bacteria. Pectinolytic bacteria, by secreting pectinases, degrade pectins contained in the medium, forming characteristic cavities in the medium that allow their identification. Within the cavities formed by pectin-metabolizing bacteria, other bacterial species may still be present, due to which the method may require additional steps to isolate SRP bacteria in the form of uniform colonies. Cases have been described in which some isolates of bacteria belonging to the Pectobacterium spp. and Dickeya spp. do not proliferate on CVP medium or do not cause cavities [1], Bacteria of the genus Pseudomonas, which may be capable of growth and cavity formation in CVP medium [3], and the difficulty of selecting an optimal source of pectins for the preparation of the medium also pose problems [4],

[0007] In addition to CVP medium, several other selective media have been developed for SRP isolation, but they are not widely used. One such medium is a modification of Miller-Schroth medium [5], in which agar is replaced by sodium polypectate, and MOPS (3-morpholinopropane-1 -sulfonic acid) buffer is used to stabilize pH. SRP colonies proliferating in this medium form pink-red-orange cavities, while the growth of other bacteria is inhibited or no characteristic cavities are observed. There are also growth media differentiating a particular type of bacteria, such as NGM medium [6], which allows differentiation of Dickeya spp., using the ability of these bacteria to produce blue pigment indigoidine, which allowed distinguishing blue colonies of Dickeya spp. from other SRP present on the medium. However, this method is sometimes ineffective, especially for samples with a high microbial background.

[0008] Other methods that allow detection of pectinolytic bacteria are serological methods based on antigen-antibody interactions. Detection is based on the interaction of poly- or monoclonal antibodies with the bacterial surface antigens — most often lipopolysaccharides (LPS) or O antigen [1]-

[0009] One of the first uses of antibodies to detect and differentiate bacteria causing soft rot and potato blackleg were agglutination and precipitation and double immunodiffusion tests [7, 8], These tests are easy to perform, including under field conditions, as they do not require the use of specialized equipment. Today, however, these methods have been replaced by other, more effective techniques.

[0010] Further methods include immunofluorescence staining (IF) and fluorescent antibody staining (FAS). These techniques use fluorophore-conjugated antibodies that bind to antigens on the surface of bacterial cell walls, allowing them to be detected using a fluorescence microscope [9],

[0011] Immunofluorescence colony staining (IFC) is a more sensitive variant of immunofluorescence staining. This method involves staining bacterial colonies rather than individual cells

[0010] ,

[0012] Currently, the most widely used serological test is the enzyme-linked immunosorbent assay (ELISA). ELISA allows detection of SRP by binding of polyclonal or monoclonal antibodies conjugated with the appropriate enzyme to specific proteins on the surface of bacterial cells. The assay itself has a low sensitivity (lower detection limit 105-106cells per gram of plant material)

[0011] , but it is possible to increase it significantly by pre-enriching the sample in selective medium before testing, which, however, increases the analysis time.

[0013] The possibility of detecting Pectobacterium spp. and Dickeya spp. using the LuminexxMAP® system has also been described

[0012] - This system, based on the principles of flow cytometry, involves the use of stained microspheres coated with, for example, primary antibodies and fluorescently labeled secondary antibodies. The technology has been compared with ELISA, with the LuminexxMAP® system proving to be more sensitive, faster and capable of simultaneous detection of multiple pathogens at the same time. However, the method is technically complex and requires the purchase of specialized equipment and reagents.

[0014] The main problem with serological tests is the specificity of the antibodies used. Polyclonal antibodies show lower specificity than monoclonal antibodies and can provide false-positive results due to the presence of common antigenic determinants among different (off-target) bacteria in the sample. Monoclonal antibodies show high specificity against a precisely defined epitope but this can be associated with false-negative test results due to the serological variability of SRP. Another obstacle is the high cost of monoclonal antibody production.

[0015] Further method that allows detection and identification of SRP is chromatographic analysis of fatty acid methyl esters (FAME) synthesized by these bacteria. This method allows detection, differentiation of Pectobacterium spp. and identification and / or characterization of unknown strains of pectinolytic bacteria based on the profiles of different fatty acids

[0013] ,

[0016] Volatile Organic Compound (VOC) analysis can also be used to detect Pectobacterium and Dickeya species

[0014] . Some of these can be linked exclusively to the presence of Pectobacterium spp.

[0015] , In 2000, an electronic test was developed; it contains three independent sensors to detect compounds produced by Pectobacterium spp. during infection of potato tubers. It was reported that the sensor system detects even latent infection of potato tubers artificially infected with Pectobacterium spp. at 85% relative humidity and 4 °C

[0016] , The use of a whole-cell bacterial biosensor allowing early identification of soft rot development in potato tubers was also described

[0017] , Detection of the bacteria is based on monitoring changes in the luminescence level of a panel of bacterial bioreporters in response to changes in the VOC profile after inoculation with Pectobacterium spp. or exposure to a potato tuber infected with Pectobacterium spp. Currently, for the detection and identification of bacteria from the SRP group, molecular methods based on the amplification of specific regions of bacterial genomic DNA are the most widely used. They allow detection of SRP in multiple samples simultaneously. These methods are characterized by high sensitivity, specificity and versatility; they can be used to detect bacteria at any level of taxonomic organization: genus, species or subspecies. A key aspect of the method is the selection of appropriate regions of the bacterial genome targeted for amplification and, consequently, the design of suitable primers for the PCR reaction. Typically, the target genes are virulence-related genes, housekeeping genes, 16S rDNA or genome fragments of unknown function, but unique to a given microorganism or microorganisms [1],

[0017] Currently, there is available a number of methods for detecting SRP based on DNA amplification such as classical PCR (polymerase chain reaction), multiplex PCR, multiplex PCR with padlock probes, real-time PCR, MLST (multilocus sequence typing) or LAMP (loop-mediated isothermal amplification). Depending on the method chosen, it is possible to identify one or multiple SRP species at the same time; it is also possible to detect SRP in real time or not, quantitatively or qualitatively. Each method differs in its specificity as to the result, the need for appropriate sample preparation, duration, and possible subsequent steps to develop the test result (e.g. agarose electrophoresis).

[0018] Tests based on amplification of the SRP-specific DNA fragments are relatively inexpensive and fast, compared to classical culture methods. They are also more specific than serological techniques. However, there are a number of limitations of this method: these tests do not distinguish between viable and non-viable bacterial cells. This can lead to false-positive test results

[0018] , False-positive results can also occur by hybridization of primers to non-SRP bacterial genomic fragments (VBNCs, sequences not cataloged in databases) similar to the selected SRP genome target sequence, which will result in non-specific amplification. Through contamination with PCR reaction inhibitors, errors in reaction optimization, reaction mixture preparation or primer design, it is possible for the target fragment of the bacterial genome to go undetected, resulting in false-negative results. Developing SRP-specific primers can also be challenging, due to the many changes in the taxonomy of this group of bacteria. These methods also require the use of specialized, usually expensive, laboratory equipment and reagents.

[0019] The next method to detect SRP is sequencing of the entire bacterial genome. Nowadays, sequencing is a highly automated process, carried out using new-generation sequencers. SRP detection by sequencing is limited to reference sequences available in specialized databases. The technique requires specialized equipment and, at the present time, is relatively expensive and time-consuming compared to other available detection methods.

[0020] There are reports of the possibility to detect Pectobacterium and Dickeya species using a method based on conductivity

[0019] , This method allowed automated detection of conductivity changes caused by the growth of Pectobacterium spp. and Dickeya spp. in extracts from potato tuber skins. The method is relatively simple and inexpensive; nevertheless, it has not been applied in routine SRP detection.

[0021] AIM OF THE INVENTION

[0022] There is a lack of diagnostic tests on the market that allow rapid detection of live bacterial cells from SRP group without the need to identify them under laboratory conditions. Therefore, the aim of the invention was to develop a portable test (usable in the field, both during cultivation and during storage) that allows the detection of live bacterial cells from SRP group, whose sensitivity and specificity match those of tests routinely performed in laboratories.

[0023] SUMMARY OF THE INVENTION

[0024] The subject matter of the present invention is a method for detecting pathogens of crops and ornamental plants, comprising the steps in which:

[0025] - a control sample is prepared by mixing an extraction buffer with a microbial medium concentrate;

[0026] - a sample of purified plant material is collected, suspended in the extraction buffer and shaken vigorously, preferably for about 5 seconds;

[0027] - the liquid from above the sample (supernatant) is transferred to a tube with the medium concentrate, which is then sealed, shaken and set aside;

[0028] - the samples are incubated for a period of 4 to 48 hours at a temperature in the range of 18-38 °C, preferably at 28 °C.

[0029] - a reading of the samples is carried out using test cassettes, characterized in that a microbial medium supplemented with a fluorescently labeled probe that allows detection of pectinolytic activity of microorganisms multiplied in the sample is used.

[0030] Preferably, the detection of pectinolytic activity in a sample is performed by separation of the enzymatic degradation products of a fluorescently labeled probe on a nitrocellulose membrane. Preferably, a microbial medium supplemented with a molecule that stimulates the expression of genes encoding pathogenicity factors, including secreted pectinolytic enzymes, such as N-( - ketocaproyl)-L-homoserine lactone, is used.

[0031] Preferably, the extraction buffer has the following composition: and the microbial medium concentrate has the following composition:

[0032] Preferably, detection of pectinolytic activity occurs during enrichment culture of plant pathogens.

[0033] Preferably, the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

[0034] Also a subject matter of the invention is a test kit for detection of pathogens of crops and ornamental plants comprising strip test reader with an incubator compartment; test cassettes with two lanes each; • extraction tubes with microbial medium concentrate;

[0035] • extraction tubes with extraction buffer, in which the extraction buffer has the following composition: and the microbial medium concentrate has the following composition:

[0036] Preferably, the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

[0037] Preferably, the strip test reader is equipped with a dedicated application to automate the analysis and interpretation of the results.

[0038] Preferably, the extraction tubes are made from soft polymer and have a dropper tip.

[0039] Preferably, the kit comprises sterile galvanized steel brushes for the extraction of plant tissue in packages that allow the tool to remain sterile.

[0040] Preferably, the kit comprises plastic sterile Pasteur pipettes, preferably individually packaged.

[0041] Further subject matter of the invention is a microbial medium with a composition:

[0042] allowing use for detecting pathogens of crops and ornamental plants.

[0043] Preferably, the medium consists of an extraction buffer with the following composition: and a microbial medium concentrate with the following composition:

[0044] Preferably, the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

[0045] Also a subject matter of the invention is the use of a microbial medium with a composition: for detecting pathogens of crops and ornamental plants. Preferably, the microbial medium consists of an extraction buffer with the following composition: and a microbial medium concentrate with the following composition:

[0046] Preferably, the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

[0047] DESCRIPTION OF THE DRAWINGS

[0048] Fig. 1. Average growth curve of bacteria from the SRP group and of the genus Pseudomonas on the microbial medium that is part of the invention. Error bars represent the standard deviation.

[0049] Fig. 2. Appearance of an example cassette after digested probe separation. Left lane: positive sample. Right lane: negative sample.

[0050] Fig. 3. Analysis of probe-on-membrane separation images obtained with the reader demonstrator during sample separation. A. Raw image of the positive sample. B. Processed image of the positive sample, presented as a graph for machine analysis. C. Raw image of the negative sample. D. Processed image of the negative sample, presented as a graph for machine analysis.

[0051] Fig. 4. Activity diagram showing the various stages of test performance.

[0052] Fig. 5. External appearance of the first reader prototype.

[0053] Fig. 6. Final version of the reader.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] The test has a form that allows the full diagnostic process to be carried out under field conditions. It allows detection of latent (asymptomatic) infections, as well as confirmation of the etiological agent in the case of symptomatic infections in diverse matrices, particularly in plant tissue (e.g., potato tubers), but also the presence of SRP in other matrices (e.g., soil or water).

[0056] A key element of the invention is a selective liquid microbial medium supplemented with a fluorescently labeled probe based on the backbone of the pectin molecule, allowing detection of pectinolytic enzyme activity in the sample.

[0057] The composition of the medium (shown in Table 1) is characterized by supporting the growth of a broad spectrum of bacterial species belonging to the SRP group, with simultaneous negative selectivity against Pseudomonas spp. and Gram-positive bacteria.

[0058] Selectivity of the medium was achieved by not using universal sources of biogenic elements (such as, for example, yeast extracts, peptone, tryptone) but compounds that do not support the growth of bacteria of the genus Pseudomonas (selected species and isolates of which can provide false-positive results in the detection of bacteria from the SRP group, such as on CVP medium [3]). Selectivity against G(+) bacteria was achieved by using crystal violet at a concentration that inhibits their growth. In addition, the microbial medium can contain compounds such as pyrroloquinoline quinone (PQQ) or an appropriate molecule with a function in quorum sensing, stimulating the expression of genes encoding pathogenicity factors, including secreted pectinolytic enzymes (e.g., N-( - ketocaproyl)-L-homoserine lactone).

[0059] The ready-to-use microbial medium is divided into 2 components: 1) extraction buffer (EB), which is mixed with the sample to allow extraction of bacterial cells from the sample and contains, among other things, detergent, buffer system and most of the inorganic components of the medium, and 2) medium concentrate (MC), which contains, among other things, organic compounds and a fluorescently labeled probe that allows detection of pectinolytic activity in the sample. MC can be in solution or lyophilizate form, where lyophilization significantly extends the shelf life and usefulness of the described invention.

[0060] Table 1. EB composition

[0061] Table 2. MC composition

[0062] Table 3. Final composition of the nutrient solution i I

[0063] The first step of the test is to obtain the test material and suspend it in the extraction buffer (EB). For matrices that allow this (e.g., water or soil), this process is carried out by adding a given volume of the test material to a tube with EB. For matrices that require more specific sampling, such as plant tissue (where, for potato tubers in particular, it is recommended to collect the tissue from the stolon point of the tuber), a suitable sterile instrument, such as a steel brush, is used, with which the material is collected, while coarsely homogenizing it, and then the collected tissue is suspended in EB.

[0064] After mixing the sample prepared in this way, EB is collected from above the material and mixed 1 :1 with the medium concentrate (MC). After shaking, the sample prepared in this way is incubated for a given time (standard 24 h) at 28 °C in order to allow the growth of bacteria from the SRP group, which produces pectinolytic enzymes. After the incubation is completed, the test result is developed.

[0065] The test is developed by applying a few drops (100-200 pL) of the medium onto a lateral flow test (LFT) cassette. The medium is applied onto a cellulose paper, acting as an absorbent pad, through which the sample migrates to a nitrocellulose membrane, on which the fluorescently labeled probe is properly separated. The probe's ability to migrate is strongly dependent on the degree of its digestion (understood as fragmentation of the molecule) as a result of the pectinolytic activity within sample.

[0066] A probe subjected to digestion undergoes fragmentation, resulting in the formation of its oligomeric fragments of sufficiently low molar mass to allow their migration and separation on a nitrocellulose membrane. The fragmented probe, during separation under specific buffer conditions, spontaneously arranges itself into a band-like pattern, which allows its detection by observation or visualization under light of a wavelength suitable for the fluorophore with which the probe is labeled (usually in the ultraviolet light range). An undigested probe, on the other hand (having an average molar mass of about 22 kDa) does not separate on the membrane, resulting in a lack of signal. The presence of a characteristic pattern (see Fig. 3) is the parameter that determines the classification of the test result as positive or negative.

[0067] An example probe could be the one described in international patent application PCT / IB2024 / 051258, which is a fluorescently labeled derivative of polygalacturonic acid or pectin, and obtained by coupling reaction of a substrate (polygalacturonic acid and / or pectin, for example, selected from such as substances with CAS numbers: 9000-69-5, 9005-88-3, 503591-24-0, 220424- 61-3, 9047-18-1, 9049-37-0, 25990-10-7, 25249-06-3, or oligo- and polymers based on structures with CAS numbers 685-73-4, 552-12-5, 14982-50-4, 6294-16-2, 9046-38-2, 25990-10-7, 9046-40-6, 18968-14-4, 9000-69-5) with a selected fluorescent dye having appropriate reactive functional groups (carboxyl groups), allowing the reaction to take place. The coupling reaction is carried out by activating the carboxyl groups of pectin / polygalacturonic acid with 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC) and incubating with the selected dye(s) in a pyridine buffer medium with a pH in the range of 4-5.

[0068] Visualization and reading of the test result take place in a dedicated device, i.e. a reader with an incubator compartment. The device is equipped with the following components:

[0069] • STM32 NUCLEO-F429ZI microcontroller,

[0070] • HC-05 Bluetooth communication module,

[0071] • heating module with thermostat,

[0072] • excitation system, consisting of 2 LEDs emitting light with a wavelength of 370 nm,

[0073] • detection system: ArduCam OV5642 5MPx camera with HQ M12x0.5 lens,

[0074] • battery module based on lithium-ion cell, providing 12V 4 A power supply,

[0075] • power port for charging the device and keeping it on mains power, • USB port for connecting the reader to a computer,

[0076] • drawer for accommodating the test cassette and reading the test,

[0077] • chamber with an aluminum heating block used to incubate collected samples,

[0078] • handle allowing the device to be carried freely.

[0079] The housing of the device was made of powder-coated rolled steel sheet to ensure resistance to external conditions and damage during handling and use under the field conditions. The construction of the device is shown in Figs. 5 and 6.

[0080] Fig. 6 shows the final version of the reader, where

[0081] A: is a view of the reader after removing the front panel and battery module.

[0082] Legend: 1. Bluetooth module; 2. STM32 microcontroller; 3. Shield connector board with signal LEDs and UV LEDs; 4. Camera; 5. Position drawer with test cassette pocket.

[0083] B: is a view of the reader after removing the front panel with battery module.

[0084] Legend: 6. Technical compartment with electronic components; 7. Optical-measuring compartment;

[0085] 8. Incubator compartment; 9. Battery module.

[0086] C: is a fragment of the rear reader panel view.

[0087] Legend: 9. Communication port with USB-B connector; 10. Power port; 11. Plastic panel for Bluetooth communication; switch visible on far right.

[0088] D: is a view of open incubator compartment.

[0089] Legend: 12. Pull-out comb to immobilize tubes during transport; 13. Aluminum heating block. Heaters and thermostat module with thermocouple not visible (mounted in heating block).

[0090] E: is a view of the open incubator compartment after installation of the front reader panel. Visible reflective layer insulating the compartment from excessive heat loss.

[0091] F: is a view of the opened slot to the optical-measuring compartment with the partially pulled-out drawer with a cassette pocket. Visible UV light reflected from the white surface of the cassette.

[0092] G: is a view of the optical-measuring compartment after removing the front reader panel. Visible camera with optimal distance set from the cassette placed in the measuring drawer.

[0093] H: is a view of the top wall of the optical-measurement compartment with the camera visible and the UV LEDs on. Legend: 14: a pair of UV LEDs placed on the sides of the camera.

[0094] The device is operated from a mobile application, which is an integral component of the test kit; it allows the user's smartphone to connect to the reader via wireless Bluetooth communication. The data acquired by the device's detector is transmitted via Bluetooth communication to the application, and from there to a serverless cloud service, where the data is analyzed and the interpreted results are returned, which the mobile application provides to the end user in the form of a report.

[0095] The application has the following functionalities:

[0096] • cell phone connection to the reader via Bluetooth,

[0097] • QR code scan of the sample,

[0098] • QR code scan of the reader,

[0099] • sample description and numbering,

[0100] • result receiving: positive, negative or inconclusive,

[0101] • result downloading in the form of a pdf report.

[0102] The backend of the application runs on the AWS Lambda platform (Amazon) and is written in the Python 3.8 programming language. Algorithms were implemented to effectively detect the digested probe signal, and to compare the acquired data to a control sample.

[0103] The analysis algorithm used receives as input the image recorded by the reader during separation. The image is analyzed for: 1. the presence of fluorescence characteristic of the labeled probe (registered as pixels with a hue characteristic of the fluorophore with which the probe is conjugated 2. arranged in the form of a pattern similar to a band. For this purpose, the color intensity is determined and presented in the form of a graph; this graph is analyzed in terms of exceeding the established threshold value of signal intensity and the presence of the aforementioned signal in the form of a peak. If all the required characteristics are met, the obtained signal is registered as a positive signal. In order to ensure adequate robustness of the detection method to possible occurring artifacts, to qualify the sample as positive, the occurrence of a signal qualified as unambiguously positive in an appropriate number of consecutive registered images is required (e.g. for registration at 0.1 Hz it is required to consider min. 4 out of 5 consecutive registered images as unambiguously positive). What is also important here is that samples are always analyzed in pairs — the control sample on the 2nd lane of the test cassette is used to determine the baseline for analysis. A visual example of the analysis is shown in Fig. 3. EXAMPLES

[0104] Example 1

[0105] The contents of the field test kit:

[0106] • strip test reader with an incubator compartment along with a dedicated application to automate the analysis and interpretation of results

[0107] • 4 test cassettes with two paths each

[0108] • 5 soft extraction tubes with dropper tip, with microbial medium concentrate (including 4 test tubes and 1 control tube)

[0109] • 4 50 mL tubes, with extraction buffer (30 mL)

[0110] • 4 sterile galvanized steel brushes, for collection of plant tissue in 15 mL plastic tubes

[0111] • 4 individually packaged, plastic sterile Pasteur pipettes.

[0112] Test performance protocol for testing (defined for a standard representative sample of potato tubers: 200 tubers in 4 subsamples of 50 tubers each):

[0113] 1. Roughly clean the potato tubers from the soil with clean water and dry with a paper towel, taking care not to damage the tubers.

[0114] 2. Wash and dry hands before proceeding to the next steps.

[0115] 3. Divide the sample of 200 tubers into 4 subsamples of 50 each.

[0116] 4. Open the first 50 mL tube with extraction buffer.

[0117] 5. Using a sterile Pasteur pipette, transfer the appropriate volume of extraction buffer into the tube with medium concentrate, labeled as the control sample, topping it up to the printed line. Close, shake and set the tube aside.

[0118] 6. Remove a clean steel brush from the plastic tube, taking care not to grab the brush by the bristles.

[0119] 7. On the potato tuber, locate the stolon point (the place where the tuber joins the vegetative part of the plant); insert ("screw") the brush into the stolon point to a depth of about 5-10 mm into the tissue. NOTE: if the tuber shows lesions, it is acceptable to take tissue from such a lesion instead of the stolon point.

[0120] 8. Remove the brush from the tuber, immerse it in the liquid in a 50 mL tube and rinse it several times in a circular motion in the buffer until the suspension of the collected tissue in the buffer is observed. 9. Using the same steel brush, repeat the process for the remaining tubers in the subsample.

[0121] 10. Cap the 50 mL tube with the tissue suspension, shake the tube vigorously for about 5 seconds to mix the contents.

[0122] 11. Using a Pasteur pipette, transfer the liquid from above the tissue to the tube with the medium concentrate, topping it up to the printed line. Close, shake and set the tube aside.

[0123] 12. Repeat steps 4 and 6-11 for tubes 2, 3 and 4.

[0124] 13. Using the dedicated application, add the test and register the prepared samples (it is suggested to scan the QR codes on the 5 tubes so prepared); add additional comments and descriptions. Confirm the addition of the samples.

[0125] 14. Start the reader; place the 5 tubes thus prepared in the aluminum heating block in the reader incubator compartment.

[0126] 15. Leave the test tubes in the incubator for 24 h*.

[0127] 16. After the incubation time has elapsed, remove the tubes from the incubator.

[0128] 17. Start the application, in the screen visible after pressing the "Start measurement" button, complete the sample identification field (by scanning the QR code).

[0129] 18. Using a dropper tip, drop 3-4 drops of sample onto the left path (marked "T"); drop 3-4 drops of liquid from the control tube onto the right path ("C").

[0130] 19. Place the cassette in the measuring drawer, push the drawer in and close the partition.

[0131] 20. Click the "Confirm" button in the dedicated application.

[0132] 21. Wait for the measurement result to be returned.

[0133] 22. Repeat steps 17-20 for subsamples 2, 3 and 4.

[0134] 23. Generate a final report with the test result.

[0135] *24 h is the default incubation time for the test. A positive result for samples heavily contaminated with SRP group bacteria can be obtained after just 4-6 h of incubation. Similarly, extending the incubation time of samples to 36-48 h allows detection of lower titers of SRP group bacteria (on the order of 10-100 viable cells in a sample). Depending on the user's expectations and the purpose of performing the test, the incubation time can be manipulated. Shortening the incubation below 4 h and extending it beyond 48 h is not recommended and may result in a false-negative or false-positive result.

[0136] Literature

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Claims

CLAIMS1. A method for detecting pathogens of crops and ornamental plants, comprising the steps in which:- a control sample is prepared by mixing extraction buffer with microbial medium concentrate;- a sample of purified plant material is collected, suspended in the extraction buffer and shaken vigorously, preferably for about 5 seconds;- the liquid from above the sample is transferred to a tube with the medium concentrate, which is then closed, shaken and set aside;- the samples are incubated for a period of 4 to 48 hours at a temperature in the range of 18-38 °C, preferably at 28 °C.- a reading of the samples is carried out using test cassettes, characterized in that a microbial medium supplemented with a fluorescently labeled probe that allows the detection of pectinolytic activity of microorganisms multiplied in the sample is used.

1. The method according to claim 1 characterized in that the detection of pectinolytic activity in a sample is performed by separating the enzymatic degradation products of a fluorescently labeled probe on a nitrocellulose membrane.

3. The method according to claims 1 or 2, characterized in that a microbial medium supplemented with a molecule that stimulates the expression of genes encoding pathogenicity factors, including secreted pectinolytic enzymes, such as N-( -ketocaproyl)-L- homoserine lactone, is used.

4. The method according to any of the claims 1-3, characterized in that the extraction buffer has the following composition:and the microbial medium concentrate has the following composition:

5. The method according to any of the claims 1-4, characterized in that the detection of pectinolytic activity occurs during enrichment culture of plant pathogens.

6. The method according to any of the claims 1-5, characterized in that the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC).

7. A test kit for detection of pathogens of crops and ornamental plants comprising• strip test reader with an incubator compartment;• test cassettes with two paths each;• extraction tubes with microbial medium concentrate;• extraction tubes with extraction buffer, characterized in that the extraction buffer has the following composition:and the microbial medium concentrate has the following composition:

8. The kit according to claim 7, characterized in that the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC).

9. The kit according to claims 7 or 8, characterized in that the strip test reader is equipped with a dedicated application to automate the analysis and interpretation of the results.

10. The kit according to any of the claims 7-9, characterized in that the extraction tubes are made from soft polymer and have a dropper tip.

11. The kit according to any of the claims 7-10, characterized in that it comprises sterile galvanized steel brushes for the extraction of plant tissue in packages that allow the tool to remain sterile.

12. The kit according to any of the claims 7-11, characterized in that it comprises plastic sterile Pasteur pipettes, preferably individually packaged.

13. A microbial medium with a composition: for detecting pathogens of crops and ornamental plants.

14. The microbial medium according to claim 13, which consists of an extraction buffer with the following composition:and a microbial medium concentrate with the following composition:

15. The microbial medium according to claims 13 or 14, characterized in that the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of carboxyl groups of pectin / polygalacturonic acid using 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC).

16. A use of microbial medium with a composition:for detecting pathogens of crops and ornamental plants.

17. The use according to claim 16, wherein the microbial medium consists of an extraction buffer with the following composition:and a microbial medium concentrate with the following composition:

18. The use according to claims 16 or 17, characterized in that the fluorescently labeled probe is a fluorescently labeled derivative of polygalacturonic acid or pectin, which is obtained by coupling reaction of polygalacturonic acid and / or pectin with a fluorescent dye through activation of the carboxyl groups of pectin / polygalacturonic acid using 1 -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDAC).

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