A Detection Method for Tropical Race 4 of the Banana Pathogen Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Fusarium wilt, the devastating disease that ruined the ‘Gros Michel’ (AAA)-based banana production in the first half of the 20th century. The occurrence of a new variant in Southeast Asia that overcomes the resistance in Cavendish clones such as ‘Grand Naine’ (AAA) is a major concern to current banana production worldwide. The threat posed by this new variant, called tropical race 4 (TR4), may be overcome by the introduction of resistant cultivars. However, the identification of new resistant sources or breeding for resistance is a long-term effort. Currently, the only option to control the disease is to avoid or reduce the spread of the pathogen by eradication of infected plants and isolation of infested plantations. This requires sensitive and highly specific diagnostics that enable early detection of the pathogen. A two-locus database of DNA sequences, from over 800 different isolates from multiple formae speciales of F. oxysporum, was used to develop a molecular diagnostic tool that specifically detects isolates from the vegetative compatibility group (VCG) 01213, which encompasses the Foc TR4 genotype. This diagnostic tool was able to detect all Foc TR4 isolates tested, while none of the Foc isolates from 19 VCGs other than 01213 showed any reaction. In addition, the developed diagnostic tool was able to detect Foc TR4 when using DNA samples from different tissues of ‘Grand Naine’ plants inoculated with TR4 isolate

Genetic Variation Among Vegetative Compatibility Groups of Fusarium oxysporum f. sp. cubense Analyzed by DNA Fingerprinting

Genetic variation within a worldwide collection of 208 isolates of Fu-sarium oxysporum f. sp. cubense, representing physiological races 1, 2, 3, and 4 and the 20 reported vegetative compatibility groups (VCGs), was analyzed using modified DNA amplification fingerprinting. Also characterized were 133 isolates that did not belong to any of the reported VCGs of F. oxysporum f. sp. cubense including race 3 isolates from a Heliconia species and isolates from a symptomatic wild banana species growing in the jungle in peninsular Malaysia. The DNA fingerprint patterns were generally VCG specific, irrespective of geographic or host origin. A total of 33 different genotypes were identified within F. oxysporum f. sp. cu-bense; 19 genotypes were distinguished among the isolates that belonged to the 20 reported VCGs, and 14 new genotypes were identified among the isolates that did not belong to any of the existing VCGs. DNA fingerprinting analysis also allowed differentiation of nine clonal lineages within F. oxysporum f. sp. cubense. Five of these lineages each contained numerous closely related VCGs and genotypes, and the remaining four lineages each contained a single genotype. The genetic diversity and geographic distribution of several of these lineages of F. oxysporum f. sp. cubense suggests that they have coevolved with edible bananas and their wild diploid progenitors in Asia. DNA fingerprinting analysis of isolates from the wild pathosystem provides further evidence for the coevolution hypothesis. The genetic isolation and limited geographic distribution of four of the lineages of F. oxysporum f. sp. cubense suggests that the pathogen has also arisen independently, both within and outside of the center of origin of the host

Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format

Soilborne Diseases and their Control

Seed and seedling diseases, root rots, and wilts are caused by a number of soilborne fungi, all of which are facultative saprophytes and can survive in soil for long periods in the absence of a susceptible host. In general, these diseases are serious yield constraints where short rotations or monoculture of legume crops are the rule. Seedling diseases and root rots are enhanced by poor seed vigor, poor seedbed preparation, and other biotic and abiotic stresses which predispose the host plant. Control of these diseases requires an integrated approach of genetic resistance/tolerance, cultural practices, appropriate seed treatments, and high seed vigor. The most economical and durable control of Fusarium wilt is to grow resistant varieties. New races of a wilt pathogen have arisen due to increased selection pressure from growing resistant varieties in short rotations but have not outpaced the development of resistant cultivars