Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains

Baek, Kwang Yeol; Lee, Hyun‐Hee; Son, Geun Ju; Lee, Pyeong An; Roy, Nazish; Seo, Young-Su; Lee, Seon-Woo

doi:10.5423/ppj.oa.11.2017.0250

Cited by 12 publications

(4 citation statements)

References 41 publications

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“…5). Previously, bacterial pathogens P. etewartii and Rathayibacter tritici were detected in grains using PCR with a minimum detection limit of 8.8 × 10 3 CFU/mL and DNA = 2 pg/μL [3]. Our result showed that the minimum detection limit of mPCR assay developed in the present study was closer to that of the previously reported PCR detection methods [3].…”

Section: Discussionsupporting

confidence: 82%

Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Yuan,

Qazi,

Huang

et al. 2024

Chem. Biol. Technol. Agric.

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Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, Ralstonia pseudosolanacearum, Pantoea ananatis, Enterobacter cloacae complex (ECC), Klebsiella pneumoniae species complex (KpSC), and K. oxytoca complex (KoC). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: pleD (GGDF structural domain-containing protein), yjfP (esterase), pelY (peripheral pectate lyase), ampD (N-acetyl-anhydromuranmyl-L-alanine amidase), and ripW (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 × 103 CFU/mL (DNA = 2.45 pg/μL). From 46 natural mulberry wilt samples, the mPCR detection rates of P. ananatis, ECC, KpSC, KoC and R. pseudosolanacearum were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was ≥ 65.5%, while the morbidity of the undetected pathogenic bacteria was ≤ 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, R. pseudosolanacearum, P. ananatis, ECC, KpSC, and KoC. Graphical abstract

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Section: Discussionsupporting

confidence: 82%

Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Yuan,

Qazi,

Huang

et al. 2024

Chem. Biol. Technol. Agric.

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“…this bacterium affects mainly the xylem vessels on the plants [57]. It causes systematic vascular bacterial wilt of plants and leaf blight [58]. In wheat the bacterium is known to cause mosaic disease symptoms.…”

Section: Discussionmentioning

confidence: 99%

Variations of the Bacterial Community in Wheat (Triticum aestivum), Oats (Avena sativa L.), Barley (Hordeum vulgare L.) and Triticale (Triticosecale) from Three Regions of the Republic of Crimea

Muvingi¹,

Slovareva²,

Yaremko³

et al. 2023

Preprint

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Russia is one of the largest cereal grain exporters in the world, churning out 34,3 million tons of export grain (wheat, barley and oats) in 2021. Plant infectious pathogens continue to be among the main factors in yield loss in the field and are a danger to the grain exporting industry's ability to expand internationally. This is primarily due to phytosanitary restrictions imposed by nations that monitor the presence and absence of certain phytopathogens in imported goods. Phytosanitary measures prevent the spread of plant pathogens, thus cutting the cost of dealing with them, once the pathogens invade new agricultural regions. This paper is devoted to the detection and identification of bacteria in samples of grain crops of three regions in The Republic of Crimea. The objects of the study were bacterial isolates from plant samples particularly wheat, oats, barley and triticale. The study was conducted in 2021. The identification of the isolates was carried out by sequencing a section 16–23S of the rRNA amplified by PCR with 8UA/519B, 27f/907r and PSf/PSr primers. Nucleotide sequences were deciphered using the Bio Edit program and compared with sequences placed in GenBank (https://blast.ncbi.nlm.nih. gov). The result of identification was considered an organism with maximum similarity. As a result, 38 samples of grain crops were collected, 95 bacterial colonies were isolated, of which 68 were identified to genus level and 22 were identified to species level. Some of the phytopathogens identified include: Agrococcus jenensis, Pseudomonas sp. and Curtobacterium sp. Some of the bacteria identified are beneficial like Ochrobactrum sp. Erwinia sp. and Pantoea sp. had a frequency of 28.95%, with Pantoea agglomerans having a frequency of 18.42%. Ochrobactrum sp. had a frequency of 10.53%. Enterococcus mundtii an frequency of 5.26%. Information about the species composition of bacteria on grain crops can be used to determine the spread of bacteria and their diagnosis and for bioinformatic analysis of genomes in search of species-specific genetic markers.

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“…The conventional PCR, multiplex PCR, and real-time quantitative PCR have been widely applied to the specific diagnosis of various Pantoea species. For instance, Baek et al ( 2018 ) used conventional PCR to diagnose Pantoea stewartii subsp. stewartii accurately in plants with a detection limit of 2 pg/μl genomic DNA.…”

Section: Introductionmentioning

confidence: 99%

Detection and Control of Pantoea agglomerans Causing Plum Bacterial Shot-Hole Disease by Loop-Mediated Isothermal Amplification Technique

et al. 2022

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Plum bacterial shot-hole caused by Pantoea agglomerans (P. agglomerans) is one of the primary bacterial diseases in plum tree planting areas, resulting in abnormal growth of plum trees and severe economic losses. Early diagnosis of P. agglomerans is crucial to effectively control plant diseases. In this study, loop-mediated isothermal amplification (LAMP) analysis for genome-specific gene sequences was developed for the specific detection of P. agglomerans. We designed the LAMP primers based on the gyrB gene of P. agglomerans. The best reaction system was 0.2 μmol·L−1 for outer primer F3/B3 and 1.6 μmol·L−1 for inner primer FIP/BIP. The LAMP reaction was optimal at 65°C for 60 min based on the color change and gel electrophoresis. This technology distinguished P. agglomerans from other control bacteria. The detection limit of the LAMP technology was 5 fg·μl−1 genomic DNA of P. agglomerans, which is 1,000 times that of the traditional PCR detection method. The LAMP technology could effectively detect the DNA of P. agglomerans from the infected leaves without symptoms after indoor inoculation. Furthermore, the LAMP technology was applied successfully to detect field samples, and the field control effect of 0.3% tetramycin after LAMP detection reached 82.51%, which was 7.90% higher than that of conventional control. The proposed LAMP detection technology in this study offers the advantages of ease of operation, visibility of results, rapidity, accuracy, and high sensitivity, making it suitable for the early diagnosis of plum bacteria shot-hole disease.

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Specific and Sensitive Primers Developed by Comparative Genomics to Detect Bacterial Pathogens in Grains

Cited by 12 publications

References 41 publications

Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Variations of the Bacterial Community in Wheat (<em>Triticum aestivum</em>), Oats (<em>Avena sativa</em> L.), Barley (<em>Hordeum vulgare</em> L.) and Triticale (<em>Triticosecale</em>) from Three Regions of the Republic of Crimea

Detection and Control of Pantoea agglomerans Causing Plum Bacterial Shot-Hole Disease by Loop-Mediated Isothermal Amplification Technique

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