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"Shining a LAMP" (Loop-Mediated Isothermal Amplification) on the Molecular Detection of Phytopathogens Phytophthora spp. and Phytophthora cactorum in Strawberry Fields.

ABSTRACT: Phytopathogenic microorganisms belonging to the genus Phytophthora have been recognized many times as causal agents of diseases that lower the yield of many plants important for agriculture. Meanwhile, Phytophthora cactorum causes crown rot and leather rot of berry fruits, mainly strawberries. However, widely-applied culture-based methods used for the detection of pathogens are time-consuming and often inaccurate. What is more, molecular techniques require costly equipment. Here we show a rapid and effective detection method for the aforementioned targets, deploying a simple molecular biology technique, Loop-Mediated Isothermal Amplification (LAMP). We optimized assays to amplify the translation elongation factor 1-α (EF1a) gene for two targets: Phytophthora spp. And Phytophthora cactorum. We optimized the LAMP on pure strains of the pathogens, isolated from organic plantations of strawberry, and successfully validated the assay on biological material from the environment including soil samples, rhizosphere, shoots and roots of strawberry, and with SYBR Green. Our results demonstrate that a simple and reliable molecular detection method, that requires only a thermoblock and simple DNA isolation kit, can be successfully applied to detect pathogens that are difficult to separate from the field. We anticipate our findings to be a starting point for developing easier and faster modifications of the isothermal detection methods and which can be applied directly in the plantation, in particular with the use of freeze-dried reagents and chemistry, allowing observation of the results with the naked eye.

SUBMITTER: Siegieda DG

PROVIDER: S-EPMC8619305 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Crown rot, caused by Phytophthora cactorum, is a devastating disease of strawberry. While most commercial octoploid strawberry cultivars (Fragaria × ananassa Duch) are generally susceptible, the diploid species Fragaria vesca is a potential source of resistance genes to P. cactorum. We previously reported several F. vesca genotypes with varying degrees of resistance to P. cactorum. To gain insights into the strawberry defence mechanisms, comparative transcriptome profiles of two resistant genotypes (NCGR1603 and Bukammen) and a susceptible genotype (NCGR1218) of F. vesca were analysed by RNA-Seq after wounding and subsequent inoculation with P. cactorum. Differential gene expression analysis identified several defence-related genes that are highly expressed in the resistant genotypes relative to the susceptible genotype in response to P. cactorum after wounding. These included putative disease resistance (R) genes encoding receptor-like proteins, receptor-like kinases, nucleotide-binding sites, leucine-rich repeat proteins, RPW8-type disease resistance proteins, and 'pathogenesis-related protein 1'. Seven of these R-genes were expressed only in the resistant genotypes and not in the susceptible genotype, and these appeared to be present only in the genomes of the resistant genotypes, as confirmed by PCR analysis. We previously reported a single major gene locus RPc-1 (Resistance to Phytophthora cactorum 1) in F. vesca that contributed resistance to P. cactorum. Here, we report that 4-5% of the genes (35-38 of ca 800 genes) in the RPc-1 locus are differentially expressed in the resistant genotypes compared to the susceptible genotype after inoculation with P. cactorum. In particular, we identified three defence-related genes encoding wall-associated receptor-like kinase 3, receptor-like protein 12, and non-specific lipid-transfer protein 1-like that were highly expressed in the resistant genotypes compared to the susceptible one. The present study reports several novel candidate disease resistance genes that warrant further investigation for their role in plant defence against P. cactorum.

Dataset Information

"Shining a LAMP" (Loop-Mediated Isothermal Amplification) on the Molecular Detection of Phytopathogens Phytophthora spp. and Phytophthora cactorum in Strawberry Fields.

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