Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China

Genome-wide identification and expression profile analysis of the NAC transcription factor family during abiotic and biotic stress in woodland strawberry

<div><p>The NAC transcription factors involved plant development and response to various stress stimuli. However, little information is available concerning the NAC family in the woodland strawberry. Herein, 37 <i>NAC</i> genes were identified from the woodland strawberry genome and were classified into 13 groups based on phylogenetic analysis. And further analyses of gene structure and conserved motifs showed closer relationship of them in every subgroup. Quantitative real-time PCR evaluation different tissues revealed distinct spatial expression profiles of the <i>FvNAC</i> genes. The comprehensive expression of <i>FvNAC</i> genes revealed under abiotic stress (cold, heat, drought, salt), signal molecule treatments (H₂O₂, ABA, melatonin, rapamycin), biotic stress (<i>Colletotrichum gloeosporioides</i> and <i>Ralstonia solanacearum</i>). Expression profiles derived from quantitative real-time PCR suggested that 5 <i>FvNAC</i> genes responded dramatically to the various abiotic and biotic stresses, indicating their contribution to abiotic and biotic stresses resistance in woodland strawberry. Interestingly, <i>FvNAC</i> genes showed greater extent responded to the cold treatment than other abiotic stress, and H₂O₂ exhibited a greater response than ABA, melatonin, and rapamycin. For biotic stresses, 3 <i>FvNAC</i> genes were up-regulated during infection with <i>C</i>. <i>gloeosporioides</i>, while 6 <i>FvNAC</i> genes were down-regulated during infection with <i>R</i>. <i>solanacearum</i>. In conclusion, this study identified candidate <i>FvNAC</i> genes to be used for the genetic improvement of abiotic and biotic stress tolerance in woodland strawberry.</p></div

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Expression profiles of <i>FvNAC</i> genes in leaves under <i>R. solanacearum</i> infection.

<p>The <i>R. solanacearum</i> infection stress was irrigating pathogenic bacteria suspension (1×10⁸ CFU) woodland strawberry seedlings.</p

Expression profiles of <i>FvNAC</i> genes in leaves under heat stress.

<p>The heat stress treatment was performed by transferring the plants to a high temperature (40°C) for 4 h following recovery. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.</p

Expression profiles of <i>FvNAC</i> genes in leaves under H₂O₂ stress.

<p>The H₂O₂ stress treatment was performed by spraying the woodland strawberry leaves with a solution containing 10 mM H₂O₂. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.</p

Expression profiles of <i>FvNAC</i> genes in leaves under <i>C. gloeosporioides</i> infection.

<p>The <i>C. gloeosporioides</i> infection stress was performed by spraying the conidiospores (1×106 conidiospores/mL) to woodland strawberry leaves surface. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.</p