Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification.

AIMS: In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 (R4) in soil. METHODS AND RESULTS: The LAMP primer set was designed based on previously verified RAPD marker sequences, and the RealAmp assay could specifically detect and distinguish R4 isolates from other related species. The detection sensitivity of the RealAmp assay was approx. 3·82 × 10(3) copies of plasmid DNA or 10(3) of spores per gram in artificially infested soil, indicating that the method is highly tolerant to inhibitor substances in soil compared to real-time PCR. Combining previously published TR4-specific detection methods with the newly established R4-specific RealAmp assay, an indirect approach to detect and differentiate ST4 isolates was achieved by comparing the detection results of R4 and TR4 simultaneously. The existence of ST4 isolates in China was subsequently confirmed through the developed approach. CONCLUSION: The developed RealAmp assay has been confirmed to be a simple, rapid and effective method to detect R4 in soil, which facilitates to further identify and distinguish ST4 isolates through the comparative analysis of detection results between TR4 and R4 simultaneously. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in China.

Revealing the different resistance mechanisms of banana 'Guijiao 9' to Fusarium oxysporum f. sp. cubense tropical race 4 using comparative proteomic analysis.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense is a worldwide devastating fungal disease in the banana industry. The disease caused by Fusarium oxysporum f. sp. cubense is becoming more and more serious. The pathogen of Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) is the most harmful one. 'Guijiao 9' is a banana cultivar with good resistance to Foc4, which is identified by resistance screening of natural variant lines. It is of great significance to explore the resistance genes and key proteins of 'Guijiao 9' for banana cultivar improvement and disease resistance breeding. In this study, iTRAQ (isobaric Tags for Relative and Absolute quantitation) was used to analyze the xylem proteomic data of banana roots from the resistant variety 'Guijiao 9' and susceptible variety 'Williams', and the differences in protein accumulation profiles between these two varieties at 24, 48, and 72 h after infection with Foc4 were compared. The identified proteins were analyzed by the protein WGCNA (Weighted Gene Correlation Network Analysis), and the differentially expressed proteins (DEPs) were verified by qRT-PCR experiments. Proteomic analysis showed that there were differences in the protein accumulation profiles of the resistant cultivar 'Guijiao 9' and the susceptible cultivar 'Williams' after infection with Foc4, and there were differences in resistance-related proteins, biosynthesis of secondary metabolites, peroxidase, and pathogenesis-related proteins. The stress response of bananas to pathogens was affected by multiple factors. Protein co-expression analysis showed that there was a high correlation between the MEcyan module and resistance, and 'Guijiao 9' had a different resistance mechanism compared with 'Williams'. SIGNIFICANCE: 'Guijiao 9' is a banana variety with good resistance to Foc4, which is identified by screening the resistance of natural variant lines in the farmland where banana plants are seriously infected by Foc4. It is of great significance to excavate the resistance genes and key proteins of 'Guijiao 9' for banana variety improvement and disease resistance breeding. The aim of this paper is to identify the proteins and related functional modules controlling the pathogenicity differences of Foc4 by comparative proteomic analysis of 'Guijiao 9', so as to understand the resistance mechanism of banana to Fusarium wilt, and offer basis for the final identification, isolation and utilization of Foc4 resistance-related genes in banana variety improvement. The research results will also provide a basis for further understanding the host-pathogen interaction and revealing the resistance mechanism of bananas.

Comparative transcriptome analysis reveals resistance-related genes and pathways in Musa acuminata banana 'Guijiao 9' in response to Fusarium wilt.

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases in bananas resulting in significant loss of Cavendish bananas production worldwide. Here we show the agronomic traits and the resistance of 'Guijiao 9' in the field trials from 2012 to 2017. And then we dissect and compare the transcriptome response from these two cultivars (cv. 'Guijiao 9' and cv. Williams) in an attempt to understand the molecular basis that contribute to the enhanced Foc tropical race 4 (Foc-TR4) resistance. 'Guijiao 9' is a Cavendish cultivar with strong resistance to Foc-TR4, which was reflected in a lower disease severity and incidence in glasshouse and field trails, when compared to the susceptible cultivar Williams. Gene expression profiles of 'Guijiao 9' and Williams were captured by performing RNA-Seq analysis on 16 biological samples collected over a six day period post inoculation with Foc-TR4. Transcriptional reprogramming in response to Foc-TR4 was detected in both genotypes but the response was more drastic in 'Guijiao 9' than in Williams. Specific genes involved in plant-pathogen interaction and defense signaling including MAPK, calcium, salicylic acid, jasmonic acid and ethylene pathways were analyzed and compared between 'Guijiao 9' and Williams. Genes associated with defense-related metabolites synthesis such as NB-LRR proteins, calmodulin-binding protein and phenylpropanoids biosynthesis genes were significantly up-regulated in 'Guijiao 9' resistant to Foc-TR4 infection. Taken together, this study highlights the important roles of plant hormone regulation and defense gene activation in mediating resistance in 'Guijiao 9'.