MNP marker site for identifying pseudomonas syringae, primer composition, reagent, kit and application

Abstract

The invention relates to the technical field of biology, and discloses MNP marker sites for identifying pseudomonas syringae, a primer composition, a reagent, a kit and application, the MNP marker composition comprises 157 marker sites from MNP-1 to MNP-157, the MNP marker site is a genome region which is screened on a pseudomonas syringae genome, has the specificity of the pseudomonas syringae and has a plurality of nucleotide polymorphisms in different pathogenic variants of the pseudomonas syringae; according to the present invention, the MNP marker site is found, the multiple primer composition is designed according to the found MNP marker site, the multiple PCR amplification can be performed by using the designed multiple primer composition, and the sequencing of the amplification product is performed by fusing with the next-generation sequencing platform, such that the high-throughput, high-efficiency and high-accuracy identification and the high-throughput, high-efficiency and high-accuracy identification and the high-throughput, high-efficiency and high-accuracy identification and the high-throughput, high-efficiency and high-accuracy identification and the high-throughput, high-efficiency and high-accuracy identification and the high-throughput, high-efficiency and high-accuracy identification and the high-efficiency and high-accuracy the technical support is provided for the problems of investigation, identification, prevention and control and the like of P.syringae pathogenic variants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an MNP marker site, primer composition, reagent, kit, and application for the identification of Pseudomonas syringae. Background Technology

[0002] *Pseudomonas syringae* (P. syringae) is a prevalent plant pathogenic bacterium containing multiple pathogenic strains that can cause various plant diseases, including kiwifruit canker, bacterial angular leaf spot in cucurbits, bacterial black spot in cruciferous vegetables, and blight halo in legumes. These diseases, characterized by their insidious nature, sudden onset, and destructiveness, have become among the most devastating diseases in agricultural production, causing significant losses to the global economy.

[0003] Traditional detection methods, such as field observation of disease symptoms and biological assays of bacteria (including bacterial isolation and purification, morphological observation, and physiological and biochemical assays), while not requiring expensive equipment, are time-consuming, labor-intensive, and have limited specificity. Although DNA sequence-based molecular detection techniques have been applied to the detection of *P. syringae*, the currently used molecular detection techniques employ a limited number of sites, have limited discrimination, and are difficult to accurately identify pathogenic species. Therefore, there is an urgent need for a highly efficient, sensitive, and accurate detection technology to achieve early and precise detection of *P. syringae* diseases and monitoring of seedling health.

[0004] Therefore, how to provide multiple novel molecular markers with high polymorphism to achieve efficient, accurate and sensitive detection of multiple markers of P. syringae in a single test, thereby enabling accurate identification of P. syringae pathogenic species, is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the problems in related technologies, this invention proposes an MNP marker site, primer composition, reagent, kit, and application for the identification of Pseudomonas syringae. It can perform qualitative identification and quantitative analysis of P. syringae, and has the effects of multi-target, high throughput, and high sensitivity, thereby overcoming the above-mentioned technical problems existing in the existing related technologies.

[0006] Therefore, the specific technical solution adopted by the present invention is as follows:

[0007] According to a first aspect of the present invention, an MNP marker combination for identifying *Pseudomonas syringae* is provided, the MNP marker combination comprising 157 marker sites from MNP-1 to MNP-157, wherein the MNP marker site is a genomic region screened on the *Pseudomonas syringae* genome that is *Pseudomonas syringae*-specific and has multiple nucleotide polymorphisms in different pathogenic species of *Pseudomonas syringae*, and the MNP marker site is at least one of the 157 marker sites from MNP-1 to MNP-157.

[0008] According to a second aspect of the present invention, a multiplex PCR primer composition for detecting the MNP marker site is provided, the multiplex PCR primer composition comprising at least one of 157 primer pairs, and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.314.

[0009] According to a third aspect of the invention, a reagent for detecting the MNP marker site is provided, the reagent comprising the multiplex PCR primer composition described above.

[0010] According to a fourth aspect of the present invention, a kit for detecting the MNP marker site is provided, the kit comprising the multiplex PCR primer composition or the reagents described herein.

[0011] Furthermore, the kit also includes a multiplex PCR premix.

[0012] According to a fifth aspect of the invention, the use of the MNP marker site, the multiplex PCR primer composition, or the reagent is provided in the preparation of a detection reagent for identifying and distinguishing *Pseudomonas syringae*.

[0013] According to a sixth aspect of the present invention, there is provided the use of the MNP marker site, the multiplex PCR primer composition, the reagent, or the kit in identifying *Pseudomonas syringae* and distinguishing pathogenic species of *Pseudomonas syringae* and plant lines or samples infected with *Pseudomonas syringae*.

[0014] Compared with the prior art, the present invention provides MNP marker sites, primer compositions, reagents, kits and applications for the identification of Pseudomonas syringae, and has the following beneficial effects:

[0015] This invention provides an MNP marker locus for identifying P. syringae. Through analysis of the genomic sequences (378 in total) of 43 pathogenic P. syringae species, 157 MNP marker loci were screened. Based on the sequence information of these 157 MNP marker loci, a multiplex primer composition was designed. Multiplex PCR amplification was performed using the designed multiplex primer composition, and the amplified products were sequenced using a next-generation sequencing platform. This allows for high-throughput, high-efficiency, and high-accuracy detection and differentiation of P. syringae and infected plants in a single operation, enabling the identification and genetic diversity differentiation of P. syringae, thus providing technical support for identification. Attached Figure Description

[0016] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0017] Figure 1 This is a schematic diagram illustrating the principle of MNP marker polymorphism according to an embodiment of the present invention;

[0018] Figure 2 This is a flowchart of the screening and primer design for MNP marker sites of P. syringae according to an embodiment of the present invention;

[0019] Figure 3 This is a flowchart of the detection process for MNP marker sites provided in an embodiment of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] According to embodiments of the present invention, an MNP marker site, primer composition, reagent, kit, and application for the identification of Pseudomonas syringae are provided.

[0022] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0023] According to a first aspect of the present invention, a set of MNP markers for the identification of Pseudomonas syringae (P. syringae) is provided, wherein the MNP marker set comprises genomic regions screened on the P. syringae genome that are specific to P. syringae and have multiple nucleotide polymorphisms within P. syringae; the MNP marker sites include at least one of 157 marker sites from MNP-1 to MNP-157.

[0024] According to a second aspect of the invention, based on a general inventive concept, a multiplex PCR primer composition for detecting the MNP marker site is also provided, the multiplex PCR primer composition comprising at least one of primer pairs 1 to 157; and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.314.

[0025] In this embodiment of the invention, the composition of the specific multiplex amplification primer composition for detecting MNP marker sites is refined, which can be used to detect P. syringae, thereby achieving high-throughput, high-efficiency, and high-accuracy detection of P. syringae pathogens.

[0026] The multiplex PCR primer composition is designed based on the above-mentioned MNP marker site. The specific composition and region of the MNP marker site can be referred to the above embodiments. Since the multiplex PCR primer composition adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0027] According to a third aspect of the invention, based on a general inventive concept, a reagent for detecting the MNP marker site is also provided, the reagent comprising the multiplex PCR primer composition.

[0028] This reagent is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0029] According to a fourth aspect of the invention, based on a general inventive concept, a kit for detecting the MNP marker site is also provided, the kit comprising the multiplex PCR primer composition or the reagent, the kit further comprising a multiplex PCR premix.

[0030] This kit is based on the above-described multiplex PCR primer composition. The specific sequence information of the multiplex PCR primer composition can be found in the above embodiments. Since this kit adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0031] According to a fifth aspect of the invention, based on a general inventive concept, the use of the MNP marker site, the primer composition, or the reagent in a detection reagent for identifying and distinguishing P. syringae is also provided.

[0032] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0033] According to a sixth aspect of the invention, based on a general inventive concept, the use of the MNP marker site, the primer composition, the reagent, or the kit in identifying pathogenic species of P. syringae and plant lines or samples infected with P. syringae is also provided.

[0034] Specifically, when used for the identification of P. syringae and plant samples infected with P. syringae, the presence of P. syringae nucleic acid in the test sample is determined after quality control based on the number of P. syringae sequencing sequences detected in the test sample and blank control, the number of MNP markers detected, and MNP marker sequence information. The quality control scheme and determination method use known P. syringae DNA as the test sample to evaluate the efficiency and accuracy of the kit in detecting P. syringae, and to formulate the quality control scheme and determination method for the kit in detecting P. syringae.

[0035] This application is based on the above-mentioned MNP marker sites. The specific composition and region of the MNP marker sites can be referred to in the above embodiments. Since this application adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0036] The technical solution of the present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0037] The creative thinking behind this invention is:

[0038] The novel molecular marker for P. syringae developed in this invention is the MNP marker site. An MNP marker site is a polymorphic marker caused by multiple nucleotides within a region of the genome. Compared with SSR and SNP markers, MNP markers have the following advantages:

[0039] (1) Abundant alleles, with 2 alleles on a single MNP marker. n Species alleles, higher than SSR and SNP, are suitable for the identification and differentiation of organisms;

[0040] (2) It has strong species differentiation ability and only requires a small number of MNP markers to achieve species identification, thus reducing the detection error rate.

[0041] The method described in this invention is based on ultramultiplex PCR combined with next-generation high-throughput sequencing technology, and can detect 157 MNP markers of P. syringae in a single test, which has the following advantages:

[0042] (1) High efficiency: Multiplex PCR is used to detect 157 MNP markers of P. syringae at once, while existing fluorescent PCR technology can detect one marker of P. syringae at once, which increases the detection efficiency by 157 times.

[0043] (2) High accuracy: Multiplex PCR is used to detect multiple markers of P. syringae at once, avoiding high false negatives caused by the failure of amplification of a single marker; the amplification products are sequenced hundreds of times using a second-generation high-throughput sequencer, and the detection results are accurate and reliable.

[0044] (3) Detection and separation of mixtures: 157 MNP markers of P. syringae are detected at one time, and P. syringae in the mixture is separated according to the detected MNP markers. Existing methods based on fluorescence PCR technology are limited by the number of fluorescence channels and background fluorescence signals, making it difficult to detect multiple markers of P. syringae at the same time.

[0045] Given the above advantages and characteristics, MNP labeling and its detection technology have potential applications in the identification and differentiation of cross-species mixtures, particularly in the detection of pathogenic P. syringae species and their early warning mechanisms. Currently, there are no reports on MNP labeling in the detection of pathogenic P. syringae species, and corresponding technologies are also lacking.

[0046] Based on this, the present invention developed MNP marker sites for P. syringae. The MNP marker sites are genomic regions with multiple nucleotide polymorphisms within the species that were screened on the P. syringae genome. The reference genome for each MNP marker site is shown in Table 1.

[0047] This invention develops a multiplex PCR primer composition for the MNP marker site of P. syringae, comprising 157 primer pairs, the nucleotide sequences of which are as shown in SEQ ID NO.1 to SEQ ID NO.314. The primers do not conflict with each other and can be efficiently amplified by multiplex PCR.

[0048] The multiplex PCR primer composition can be used as a detection kit for the MNP marker sites of P. syringae. At the same time, the kit provided by the present invention can accurately identify P. syringae and distinguish different genetic backgrounds.

[0049] In the reproducibility test of this invention, the logarithm of the difference in major MNP marker genotypes between different libraries and between different library construction batches for each sample was 0, the reproducibility r = 100%, and the accuracy a = 100%.

[0050] The MNP markers and the kit described in this invention have high specificity for detecting P. syringae.

[0051] Example 1

[0052] Screening of MNP marker sites for P. syringae and design of primers for multiplex PCR amplification:

[0053] Screening of MNP marker sites for P. syringae:

[0054] like Figure 1 As shown in Table 1, based on 235 publicly available genome sequences of P. syringae and 143 genome sequences of other pathogenic species of Ps, a total of 157 MNP marker sites were screened through sequence alignment.

[0055] Table 1. MNP marker sites and the starting positions of multiplex PCR amplification primers on the reference sequence.

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] The specific steps for the above screening are as follows:

[0065] The genome sequence (NZ_CP032631.1) of a representative strain of P. syringae was selected as the reference genome, and the other genome sequences were compared with the reference genome to obtain the single nucleic acid polymorphism sites of the pathogenic species of P. syringae.

[0066] On the reference genome, a window of 80bp to 300bp is used for translation with a step size of 30kb to screen for regions with multiple candidate MNP marker sites. The candidate MNP marker site regions are required to contain ≥1 of the single nucleotide variant sites and the single nucleotide polymorphism sites are not present on the sequences at both ends of the 50bp.

[0067] Sites with high DP (discrimination) values ​​in the candidate polynucleotide polymorphism (PNP) site region were selected; the selection criteria included:

[0068] DP = d / t;

[0069] In the formula, t is the number of comparisons when all genomes are compared pairwise in the candidate polynucleotide polymorphism site region, and d is the number of genome pairs with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.

[0070] Other step sizes can also be used in the window translation stage, not limited to 30kb, but the use of 30kb in this invention is beneficial for comprehensive screening of P. syringae.

[0071] Design of primers for multiplex PCR amplification:

[0072] like Figure 2As shown, multiplex PCR amplification primers for the candidate MNP marker were designed using primer design software. The primer design followed the principle that primers do not interfere with each other, and all primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.

[0073] Evaluation of the detection efficiency of primer combinations:

[0074] DNA samples of *P. syringae*, native to my country, were prepared with genome copy numbers of 1 copy / reaction, 10 copies / reaction, 100 copies / reaction, and 1000 copies / reaction. An equal volume of sterile water was used as a blank control. Three replicate libraries were analyzed for each sample over three consecutive days, resulting in nine sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and thresholds for contamination in the quality control system and detection of the target *P. syringae* were established. The detection procedure for MNP marker sites is as follows: Figure 3 As shown.

[0075] Sensitivity and stability: As shown in Table 2, an average of 22 sites were detected at 1 copy / reaction; an average of 64 sites were detected at 10 copies / reaction; and in the detection of positive samples at 100 copies / reaction and 1000 copies / reaction, all 157 MNP marker sites of P. syringae were detected, and the sequence was specifically aligned with the reference sequence of P. syringae. This indicates that the primers and detection method have technical stability, high specificity, and sensitivity as low as 1 copy / reaction for detecting the bacteria.

[0076] Example 2

[0077] Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is as follows:

[0078] Performance evaluation and threshold setting for MNP markers and primers in identifying P. syringae:

[0079] The detection of P. syringae exhibits technical stability, high specificity, and sensitivity as low as 1 copy / reaction.

[0080] DNA samples of *P. syringae*, native to my country, were prepared with genome copy numbers of 1 copy / reaction, 10 copies / reaction, 100 copies / reaction, and 1000 copies / reaction. An equal volume of sterile water was used as a blank control. Three replicate libraries were analyzed for each sample over three consecutive days, resulting in nine sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and thresholds for contamination in the quality control system and detection of the target *P. syringae* were established. The detection procedure for MNP marker sites is as follows: Figure 3 As shown.

[0081] Table 2 shows the analysis of the detection sensitivity and stability of the MNP labeling detection kit.

[0082]

[0083] As shown in Table 2, an average of 22 sites were detected at 1 copy / reaction; an average of 64 sites were detected at 10 copies / reaction; and in the detection of positive samples at 100 copies / reaction and 1000 copies / reaction, all 157 MNP marker sites of P. syringae were detected, and the sequence specifically aligned with the reference sequence of P. syringae, indicating that the primers and detection method have technical stability, high specificity, and sensitivity as low as 1 copy / reaction for detecting the bacteria.

[0084] Evaluation of the reproducibility and accuracy of the MNP marker detection kit for detecting P. syringae:

[0085] Based on whether the genotypes of common detection sites in the three replicates can be reproduced, the reproducibility and accuracy of the MNP marker detection method for detecting P. syringae were evaluated. Specifically, the genotypes of each MNP site generated from nine sets of data from 1000 copies / reaction positive samples were compared, and the results are shown in Table 3.

[0086] Table 3. Reproducibility and accuracy assessment of MNP marker detection kits for genotype detection.

[0087] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a S-1 S-2 157 157 100％ 100％ S-1 S-3 157 157 100％ 100％ S-1 S-4 157 157 100％ 100％ S-1 S-5 157 157 100％ 100％ S-1 S-6 157 157 100％ 100％ S-1 S-7 157 157 100％ 100％ S-1 S-8 157 157 100％ 100％ S-1 S-9 157 157 100％ 100％

[0088] As shown in Table 3, the number of MNP markers with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of the number of reproducible loci to the number of shared loci. In the reproducibility test of this invention, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0089] In both 100 copies / reaction and 1000 copies / reaction positive samples, 157 MNP markers of P. syringae could be reliably detected, while a maximum of 4 MNP markers were detected in the blank control. Therefore, the criterion for determining P. syringae positivity in this invention is: when no less than 22 MNP marker sites of P. syringae are detected in the sample, and the sequence clustering is aligned to the P. syringae reference genome, the nucleic acid of P. syringae is determined to be detected in the sample.

[0090] Example 3

[0091] Application of MNP marker sites, kits, and methods in the identification of P. syringae

[0092] Five nucleic acid samples, named S1-S5, of kiwifruit infected with Pseudomonas syringaepv. actinidiae (Psa), provided by the Guangxi Zhuang Autonomous Region Institute of Special Crops, were tested using an MNP marker site detection kit. The results are shown in Table 4.

[0093] Table 4. Detection and analysis of P. syringae in the samples.

[0094]

[0095]

[0096] As shown in Table 4, the kit and method detected the MNP marker site of Actinidia kiwifruit ulcerans in kiwifruit leaf samples infected with kiwifruit ulcer disease.

[0097] As shown in Table 4, the kit and method described herein can accurately separate multiple MNP markers in a single reaction, while the method based on fluorescent PCR for detecting one marker in a single reaction requires 157 tests to achieve the expected detection effect. This demonstrates the high efficiency and accuracy of the kit and method described herein in the identification of P. syringae.

[0098] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0099] (1) The 157 highly polymorphic MNP marker sites provided in this embodiment of the invention have high specificity in identifying P. syringae.

[0100] (2) The high polymorphism multiplex PCR amplification primer composition provided in this embodiment of the invention can amplify 157 MNP marker sites in one reaction and integrate with a second-generation sequencing platform to sequence the amplification products, thereby achieving the differentiation of 157 MNP marker sites in one reaction.

[0101] The joint detection and differentiation of 157 MNP marker sites provides technical support for the efficient and accurate identification and differentiation of P. syringae.

[0102] (3) The kit for high polymorphic MNP marker sites provided in this embodiment of the invention can detect all 157 MNP marker sites in a single reaction. In the reproducibility test, all 157 marker sites were stably detected, demonstrating the high stability of the kit. The logarithm of the difference in major MNP marker genotypes between different libraries and different batches of libraries detected by the kit was 0, with a reproducibility rate r = 100% and an accuracy a = 100%, indicating the high accuracy and stability of the kit in complex templates.

[0103] (4) The primer composition provided in the embodiments of the present invention can be used to detect samples by high-throughput sequencing. By adding a unique tag to each sample, it is possible to detect hundreds or thousands of samples at once, and the detection efficiency can be further improved.

[0104] (5) The application provided in the embodiments of the present invention, compared with the traditional fluorescent PCR method which requires parallel experiments of standard samples to detect fluorescent signals, only requires high-throughput sequencing to detect the sample, and then identification of P. syringae by the detected MNP-labeled base sequence. There is no need for parallel detection of standard samples, the operation is simple and the data processing is simple, which improves the detection efficiency.

[0105] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An MNP marker combination for the identification of *Pseudomonas syringae*, characterized in that, The MNP marker combination includes 157 marker sites from MNP-1 to MNP-157, wherein the MNP marker sites are genomic regions screened on the *Pseudomonas syringae* genome that are *Pseudomonas syringae*-specific and have multiple nucleotide polymorphisms in different pathogenic species of *Pseudomonas syringae*.

2. A multiplex PCR primer composition for detecting the MNP marker site as described in claim 1, characterized in that, The multiplex PCR primer composition comprises 157 primer pairs, and the nucleotide sequences of the 157 primer pairs are shown in SEQ ID NO.1-SEQ ID NO.

314.

3. A reagent for detecting the MNP marker site as described in claim 1, characterized in that, The reagent includes the multiplex PCR primer composition as described in claim 2.

4. A kit for detecting the MNP marker site as described in claim 1, characterized in that, The kit includes the multiplex PCR primer composition as described in claim 2 or the reagent as described in claim 3.

5. The reagent kit according to claim 4, characterized in that, The kit also includes a multiplex PCR premix.

6. The use of an MNP marker site as described in claim 1, a multiplex PCR primer composition as described in claim 2, or a reagent as described in claim 3 in the preparation of a detection reagent for identifying and differentiating *Pseudomonas syringae*.

7. The use of an MNP marker site as described in claim 1, a multiplex PCR primer composition as described in claim 2, a reagent as described in claim 3, or a kit as described in any one of claims 4-5 in identifying *Pseudomonas syringae* and distinguishing pathogenic species of *Pseudomonas syringae* and plant lines or samples infected with *Pseudomonas syringae*.

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