Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR
Bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) and bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc) are two important bacterial diseases of rice. Identification of Xoo and Xoc belonging to the same species is critical for quarantine and cont...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Zhejiang University Press
2023-02-01
|
| Series: | 浙江大学学报. 农业与生命科学版 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2021.12.271 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849247335990689792 |
|---|---|
| author | ZHANG Jiannan WANG Yiming ZHANG Jiejing CHEN Lei LUO Jinyan YI Jianping LI Bin AN Qianli |
| author_facet | ZHANG Jiannan WANG Yiming ZHANG Jiejing CHEN Lei LUO Jinyan YI Jianping LI Bin AN Qianli |
| author_sort | ZHANG Jiannan |
| collection | DOAJ |
| description | Bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) and bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc) are two important bacterial diseases of rice. Identification of Xoo and Xoc belonging to the same species is critical for quarantine and control the diseases. Here, we found that the gene fhuE encoding ferric-rhodotorulic acid/ferric-coprogen receptor in Xoo and Xoc became pseudogenes after partial deletion during evolution. We designed Xoc-specific primers XocFhuE-F (5<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M5"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>-ATCGAACGATGTCACCAGGG-3<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M6"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>) and XocFhuE-R (5<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M7"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>-AGAAACGTGCGGCCAGATAA-3<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M8"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>) targeting the sequences present in the Xoc fhuE pseudogene but absent in the Xoo fhuE pseudogene. Polymerase chain reaction (PCR) amplification produced only a 159 bp DNA fragment from only Xoc strains using the primers XocFhuE-F/XocFhuE-R. Based on the Xoc-specific primers, we developed SYBR Green quantitative real-time PCR (qPCR) and EvaGreen droplet digital PCR (dPCR) methods to detect and identify Xoc. In a 20 μL reaction system with 1 μL of template, the detection limit of qPCR on Xoc was 1.6×10<sup>4</sup> CFU/mL in bacterial suspension and 1.2×10<sup>3</sup> CFU/seed in rice seeds; the detection limit of dPCR on Xoc was 1.6×10<sup>3</sup> CFU/mL in bacterial suspension and 1.2×10<sup>2</sup> CFU/seed in rice seeds. In conclusion, SYBR Green qPCR and EvaGreen dPCR based on the Xoc-specific primers XocFhuE-F/XocFhuE-R provide effective methods for quarantine of Xoc and monitoring of the bacterial leaf streak disease of rice. |
| format | Article |
| id | doaj-art-93dee08bea404636b8d6ae8e2aea3e95 |
| institution | Kabale University |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2023-02-01 |
| publisher | Zhejiang University Press |
| record_format | Article |
| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-93dee08bea404636b8d6ae8e2aea3e952025-08-20T03:58:14ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552023-02-0149556410.3785/j.issn.1008-9209.2021.12.27110089209Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCRZHANG JiannanWANG YimingZHANG JiejingCHEN LeiLUO JinyanYI JianpingLI BinAN QianliBacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) and bacterial leaf streak of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc) are two important bacterial diseases of rice. Identification of Xoo and Xoc belonging to the same species is critical for quarantine and control the diseases. Here, we found that the gene fhuE encoding ferric-rhodotorulic acid/ferric-coprogen receptor in Xoo and Xoc became pseudogenes after partial deletion during evolution. We designed Xoc-specific primers XocFhuE-F (5<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M5"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>-ATCGAACGATGTCACCAGGG-3<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M6"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>) and XocFhuE-R (5<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M7"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>-AGAAACGTGCGGCCAGATAA-3<inline-formula><alternatives><mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M8"><mml:mi mathvariant="normal">´</mml:mi></mml:math><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/9EF5767D-901A-4e03-A6D2-DE2B4A8111EB-M005.jpg"/></alternatives></inline-formula>) targeting the sequences present in the Xoc fhuE pseudogene but absent in the Xoo fhuE pseudogene. Polymerase chain reaction (PCR) amplification produced only a 159 bp DNA fragment from only Xoc strains using the primers XocFhuE-F/XocFhuE-R. Based on the Xoc-specific primers, we developed SYBR Green quantitative real-time PCR (qPCR) and EvaGreen droplet digital PCR (dPCR) methods to detect and identify Xoc. In a 20 μL reaction system with 1 μL of template, the detection limit of qPCR on Xoc was 1.6×10<sup>4</sup> CFU/mL in bacterial suspension and 1.2×10<sup>3</sup> CFU/seed in rice seeds; the detection limit of dPCR on Xoc was 1.6×10<sup>3</sup> CFU/mL in bacterial suspension and 1.2×10<sup>2</sup> CFU/seed in rice seeds. In conclusion, SYBR Green qPCR and EvaGreen dPCR based on the Xoc-specific primers XocFhuE-F/XocFhuE-R provide effective methods for quarantine of Xoc and monitoring of the bacterial leaf streak disease of rice.https://www.academax.com/doi/10.3785/j.issn.1008-9209.2021.12.271bacterial leaf streak of ricebacterial leaf blight of rice<italic>Xanthomonas oryzae</italic> conserved signature proteinferric-coprogen receptor |
| spellingShingle | ZHANG Jiannan WANG Yiming ZHANG Jiejing CHEN Lei LUO Jinyan YI Jianping LI Bin AN Qianli Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR 浙江大学学报. 农业与生命科学版 bacterial leaf streak of rice bacterial leaf blight of rice <italic>Xanthomonas oryzae</italic> conserved signature protein ferric-coprogen receptor |
| title | Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR |
| title_full | Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR |
| title_fullStr | Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR |
| title_full_unstemmed | Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR |
| title_short | Detection and identification of Xanthomonas oryzae pv. oryzicola using quantitative real-time PCR and digital PCR |
| title_sort | detection and identification of xanthomonas oryzae pv oryzicola using quantitative real time pcr and digital pcr |
| topic | bacterial leaf streak of rice bacterial leaf blight of rice <italic>Xanthomonas oryzae</italic> conserved signature protein ferric-coprogen receptor |
| url | https://www.academax.com/doi/10.3785/j.issn.1008-9209.2021.12.271 |
| work_keys_str_mv | AT zhangjiannan detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT wangyiming detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT zhangjiejing detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT chenlei detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT luojinyan detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT yijianping detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT libin detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr AT anqianli detectionandidentificationofxanthomonasoryzaepvoryzicolausingquantitativerealtimepcranddigitalpcr |