Conventional approaches for identifying the causal agent of plant diseases have traditionally involved the utilization of visual symptom assessment, microscopy techniques, and culturing methods (Ward et al. 2004). Nevertheless, reliance on these straightforward, cost-effective, and time-consuming methods can sometimes lead to erroneous conclusions, particularly when there are striking similarities between disease symptoms and the morphological characteristics of pathogens. This inherent limitation highlights the heavy dependence of these methods on prior knowledge and experimental expertise (Nezhad 2014). Up to now,
The rapid advancements in molecular biotechnology have carried out the way for the application of rapid and dependable detection methods, including PCR (Ward et al. 2004). The development of race-specific molecular markers with PCR-based techniques is very effective, quick, and simple. Molecular identification of pathogens through PCR saves time (within a few hours) and is less laborious for detecting plant pathogenic bacterial races (Song et al. 2014). The emergence of Next-Generation Sequencing (NGS), has introduced a pioneering approach to diagnostics, and methods for detecting and identifying phytopathogens (Chalupowicz et al. 2019). These advancements have paved the way for DNA-based NGS, which encompasses many steps including variant/mutation annotation and interpretation (Qin 2019). Various variations, including single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs), and structural variations can be discovered using the population genomics datasets based on NGS (Potgieter et al. 2020). These variations can be exploited to design molecular markers and can be used for plant pathogen diagnosis (Afrin et al. 2020; Rubel et al. 2019a). Many researchers have used PCR-based molecular markers to identify bacterial and fungal pathogens. Two (Sequence Characterized Amplified Region) SCAR markers were developed to detect the fungus
The 16 bacterial strains were used in this study, including nine
Table 1 . List of bacterial strains used in this study.
SL. | Bacterial Strains | Races | Host | Country | Collection Year | References |
---|---|---|---|---|---|---|
1 | 1 | US | 2017 | Vicente et al. (2001) | ||
2 | 2 | US | 2017 | |||
3 | 3 | UK | 2017 | |||
4 | 4 | UK | 2017 | |||
5 | 5 | Australia | 2017 | |||
6 | 6 | Portugal | 2017 | |||
7 | 7 | UK | 2017 | |||
8 | 8 | Spain | 2017 | Lema et al. (2012) | ||
9 | 9 | - | UK | 2022 | NCPPB | |
10 | - | UK | 2017 | Vicente et al. (2001) | ||
11 | 2 | UK | 2017 | |||
12 | - | South Korea (Suwon) | 2017 | KACC | ||
13 | - | South Korea (Yongin) | 2017 | |||
14 | - | - | South Korea | 2017 | ||
15 | - | New Zealand | 2016 | ICMP | ||
16 | - | New Zealand | 2016 |
Note: NCPPB-The National Collection of Plant Pathogenic Bacteria, KACC- Korean Agriculture Culture Collection, Jeollabuk-do, Korea; ICMP-International Collection of Microorganisms from Plants, Auckland, New Zealand; HRI-W-Horticulture Research International, Wellesbourne, UK
The bacterial DNA was extracted from 16 bacterial strains using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. The concentration and quality of all the DNA after extraction were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA). Then, the DNA was stored at -20℃ for further experiments.
The whole genome sequences of
The flanking DNA sequences near the variant regions were used to design primers using Primer3 (https://primer 3.ut.ee/), thereafter twenty primer pairs were designed (Supplementary Table S1). PCR was performed with a 10 μL reaction mixture containing 1 μL (30 ng μL-1) of DNA, 0.5 μL of each 10 pmol forward and reverse primers, 5 μL of 2X Prime Taq Premix (GenetBio, Daejeon, Korea) and 3 μL of sterile distilled water. The PCR conditions for race 4 and race 9-specific markers were adjusted with denaturation at 94℃ for 2 minutes followed by 30 cycles (94℃ for 20 seconds, 70℃ for 30 seconds, and 72℃ for 20 seconds) and terminated by a final elongation at 72℃ for 2 minutes, 25℃ overnight (Supplementary Table S1). The PCR products were analyzed with gel electrophoresis using 1.5% agarose at 100 V for 30 minutes and visualized with a gel documentation system under UV light (320 nm). The size of PCR products was determined using HiQ 100 bp DNA ladder. Additionally, reported primers for specific amplification of
To evaluate the sensitivity and specificity of 'XccR9-2F2-2R1' primer, 30 ng μL-1 DNA of races 4 and 9 was taken and serially diluted up to 10-4 dilution (30 ng μL-1, 3 ng μL-1, 0.3 ng μL-1, 0.03 ng μL-1, 0.003 ng μL-1). Thereafter, 1 μL of DNA from each dilution was used for PCR amplification using the PCR conditions described above.
The PCR amplicons of
The susceptible cabbage lines were artificially inoculated with strains of
The comparison of whole genome sequences, including
In this study, we developed a marker for early and quick detection of
We further conducted tests to evaluate the efficiency and sensitivity of the newly developed marker 'XccR9-2F2-2R1'. Remarkably, this marker successfully detected DNA from
Cloning and sequencing of the PCR amplicons produced by the 'XccR9-2F2-2R1' primer in races 4 and 9 DNA revealed a 190 bp deletion in race 9 when compared to race 4. Therefore, this marker can be used proficiently to differentiate race 4 and race 9 with amplicon sizes of 830 bp and 1080 bp, respectively (Supplementary Figs. S2a, S2b).
The cloned and sequenced amplicons were subjected to Open Reading Frame (ORF) prediction using NCBI tool ORF Finder. The number of ORFs found in race 9 and race 4 cloned fragments was six and nine, respectively. The largest ORF found was 843 nucleotides and 630 nucleotides in length and encoded for 280 amino acids and 206 amino acids in race 4 and race 9, respectively (Supplementary Figs. S2c, S2d). This protein has significant homology with the protein-encoding IS5 transposase family, but the protein sequence of the race 9 amplicon has a 71 bp deletion when compared to the protein sequence of the race 4 amplicon (Fig. S2d).
A bio-PCR assay was conducted to validate the competency of the developed markers in detecting the presence of
The establishment of races and differentiation of plant pathogens is associated with the presence of
Moreover, PCR-based molecular marker techniques have been successfully employed for the detection of bacterial and fungal pathogens.
PCR-based methods have also proven effective in identifying other pathogens, including race 3 of
Insertion sequences (IS) and transposons play a significant role in bacterial pathogenicity and evolution, including
In conclusion, this study developed a molecular marker-based PCR amplification for the specific identification of races 4 and 9 through the realignment of whole genome sequences of
This study was supported by grants from Ministry of Agriculture, Food and Rural Affairs (MAFRA) (322059- 03-2-HD050) and Regional Innovation Strategy (RIS) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-002). We would thank Dr. Pilar Soengas, Department of Plant Genetics, Spain for providing
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