Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR
<i>Pectobacterium brasiliense</i> (<i>Pbr</i>) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method fo...
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| Format: | Article |
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MDPI AG
2023-11-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/11/2808 |
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| author | Junhui Li Ruxing Chen Ruwei Yang Xinchen Wei Hua Xie Yanxia Shi Xuewen Xie Ali Chai Tengfei Fan Baoju Li Lei Li |
| author_facet | Junhui Li Ruxing Chen Ruwei Yang Xinchen Wei Hua Xie Yanxia Shi Xuewen Xie Ali Chai Tengfei Fan Baoju Li Lei Li |
| author_sort | Junhui Li |
| collection | DOAJ |
| description | <i>Pectobacterium brasiliense</i> (<i>Pbr</i>) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for <i>Pbr</i> by screening specific and sensitive primers based on the <i>glu</i> gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y = −3.391x + 36.28; R<sup>2</sup> = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 10<sup>2</sup> CFU·mL<sup>−1</sup>. The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 10<sup>2</sup> to 10<sup>4</sup> CFU·g<sup>−1</sup> viable <i>Pbr</i> bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by <i>Pbr</i> is crucial. |
| first_indexed | 2024-03-09T16:35:46Z |
| format | Article |
| id | doaj.art-f2826a490efa46c7a68e8ba48218eb42 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T16:35:46Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-f2826a490efa46c7a68e8ba48218eb422023-11-24T14:57:20ZengMDPI AGMicroorganisms2076-26072023-11-011111280810.3390/microorganisms11112808Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCRJunhui Li0Ruxing Chen1Ruwei Yang2Xinchen Wei3Hua Xie4Yanxia Shi5Xuewen Xie6Ali Chai7Tengfei Fan8Baoju Li9Lei Li10State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaComprehensive Experimental Farm, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaBeijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaState Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China<i>Pectobacterium brasiliense</i> (<i>Pbr</i>) has caused significant economic losses in major vegetable production areas in Northern China by causing bacterial soft rot in cash crops such as potatoes and cucumbers. This study aimed to establish a PMA-qPCR detection method for <i>Pbr</i> by screening specific and sensitive primers based on the <i>glu</i> gene and the conserved region of the 23S rRNA gene. Based on the optimized PMA pretreatment conditions, a standard curve was designed and constructed for PMA-qPCR detection (y = −3.391x + 36.28; R<sup>2</sup> = 0.99). The amplification efficiency reached 97%, and the lowest detection limit of viable cells was approximately 2 × 10<sup>2</sup> CFU·mL<sup>−1</sup>. The feasibility of the PMA-qPCR method was confirmed through a manually simulated viable/dead cell assay under various concentrations. The analysis of potato tubers and cucumber seeds revealed that nine naturally collected seed samples contained a range from 10<sup>2</sup> to 10<sup>4</sup> CFU·g<sup>−1</sup> viable <i>Pbr</i> bacteria. Furthermore, the system effectively identified changes in the number of pathogenic bacteria in cucumber and potato leaves affected by soft rot throughout the disease period. Overall, the detection and prevention of bacterial soft rot caused by <i>Pbr</i> is crucial.https://www.mdpi.com/2076-2607/11/11/2808<i>Pectobacterium brasiliense</i>bacterial soft rotPMA-qPCRdetectionviable cell |
| spellingShingle | Junhui Li Ruxing Chen Ruwei Yang Xinchen Wei Hua Xie Yanxia Shi Xuewen Xie Ali Chai Tengfei Fan Baoju Li Lei Li Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR Microorganisms <i>Pectobacterium brasiliense</i> bacterial soft rot PMA-qPCR detection viable cell |
| title | Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR |
| title_full | Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR |
| title_fullStr | Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR |
| title_full_unstemmed | Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR |
| title_short | Rapid Detection and Quantification of Viable Cells of <i>Pectobacterium brasiliense</i> Using Propidium Monoazide Combined with Real-Time PCR |
| title_sort | rapid detection and quantification of viable cells of i pectobacterium brasiliense i using propidium monoazide combined with real time pcr |
| topic | <i>Pectobacterium brasiliense</i> bacterial soft rot PMA-qPCR detection viable cell |
| url | https://www.mdpi.com/2076-2607/11/11/2808 |
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