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Development of a Genome-Informed Protocol for Detection of <i>Pseudomonas amygdali</i> pv. <i>morsprunorum</i> Using LAMP and PCR
oleh: Daniela Díaz, Alan Zamorano, Héctor García, Cecilia Ramos, Weier Cui, Claudia Carreras, María Francisca Beltrán, Boris Sagredo, Manuel Pinto, Nicola Fiore
| Format: | Article |
|---|---|
| Diterbitkan: | MDPI AG 2023-12-01 |
Deskripsi
One of the causal agents of bacterial canker is <i>Pseudomonas amygdali</i> pv. <i>morsprunorum</i>—Pam (formerly <i>Pseudomonas syringae</i> pv. <i>morsprunorum</i>). Recently detected in Chile, Pam is known to cause lesions in the aerial parts of the plant, followed by more severe symptoms such as cankers and gummosis in the later stages of the disease. This study presents the design of PCR and LAMP detection methods for the specific and sensitive identification of <i>Pseudomonas amygdali</i> pv. <i>morsprunorum</i> (Pam) from cherry trees. Twelve <i>Pseudomonas</i> isolates were collected, sequenced, and later characterized by Multi-locus Sequence Analysis (MLSA) and Average Nucleotide Identity by blast (ANIb). Three of them (11116B2, S1 Pam, and S2 Pam) were identified as <i>Pseudomonas amygdali</i> pv. <i>morsprunorum</i> and were used to find specific genes through RAST server, by comparing their genome with that of other <i>Pseudomonas</i>, including isolates from other Pam strains. The effector gene <i>HopAU1</i> was selected for the design of primers to be used for both techniques, evaluating sensitivity and specificity, and the ability to detect Pam directly from plant tissues. While the PCR detection limit was 100 pg of purified bacterial DNA per reaction, the LAMP assays were able to detect up to 1 fg of purified DNA per reaction. Similar results were observed using plant tissues, LAMP being more sensitive than PCR, including when using DNA extracted from infected plant tissues. Both detection methods were tested in the presence of 30 other bacterial genera, with LAMP being more sensitive than PCR.