RESUMO
Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is a serious disease that threatens banana production worldwide. It is a long-standing problem in Hawaii, but there was little knowledge of the causal pathogen. We isolated a strain of Foc, named Foc-UH, from a field experiencing the disease epidemic in Hawaii. Infection assays of a diverse panel of 26 banana clones, including varieties used for differentiating pathogen races and fruit production, revealed Foc-UH has a race 1 pathogenic phenotype with an intermediate race 2 virulence, and revealed the differential resistance of varieties to infection. Separate phylogenetic analyses using the barcoding regions of three nuclear genes, seven complete nuclear genes, and single nucleotide polymorphisms within conserved whole genome protein coding sequences, placed Foc-UH into recently proposed taxonomic frameworks relevant to Foc and the Fusarium oxysporum species complex. Screening of the 99.7% complete draft genome identified five secreted in xylem (SIX) gene homologs, including SIX1d, SIX1f, SIX9a, SIX9b, and SIX13a. This profile is similar to that of several race 1 isolates except the absence of SIX4 and SIX6. Foc-UH was morphologically dissimilar to the nearest related isolates. Altogether, this study identified a unique isolate that causes banana Fusarium wilt, which represents the first characterization of the causal pathogen in Hawaii. The findings and the genomic resources generated in this study are expected to guide banana breeding and cultivar deployment in Hawaii and beyond, and contribute to further understanding of the pathogenicity and evolutionary systematics of Foc.
RESUMO
Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18-20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.
