García-Rodríguez, Raymond O.

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    Pathogenic, phenotypic and genetic characterization of Fusarium oxysporum f. sp. cubense isolates afeccting banana (Musa spp.) cultivars in Puerto Rico
    (2016) García-Rodríguez, Raymond O.; Rivera-Vargas, Lydia I.; College of Agricultural Sciences; Irish, Brian M.; Giraldo, Martha C.; Róman Paoli, Elvin; Department of Crops and Agro-Environmental Sciences; Palomera, Rogelio
    Panama disease, or Fusarium wilt as it is also know, caused by Fusarium oxysporum f. sp. cubense (FOC) is one of the most destructive and widely disseminated diseases of banana (Musa spp.). The objectives of this study were to (1) assemble a collection of FOC isolates from symptomatic banana plants present in Puerto Rico; (2) characterize the fungal isolates in terms of their pathogenicity, phenotype and molecularly; and (3) screen isolates by the vegetative compatibility groups (VCG) using known testers. A total of 28 monosporic isolates were recovered from symptomatic banana plants from the municipalities of Isabela, Aguada, Mayaguez and Gurabo. Tissue-culture-derived plantlets of cultivars ‘2-R-2, 500’ (Gros Michel, race 1 differential), ‘Dole’ (Bluggoe, race 2 differential) and ‘5-A’ (Cavendish, race 4 differential) were used in pathogenicity assays. Inoculations were carried out by immersion of roots in a 106 conidia mL-1 aqueous suspension. Two months post-inoculation, disease severity was measured on internal rhizome discoloration and symptoms. The isolates were characterized phenotypically based on colony appearance and microscopic morphological traits produced on potato dextrose agar (PDA) and carnation leaf agar (CLA). For VCG complementation tests, nitrate non-utilizing (nit) mutants from wild-type isolates were generated in PDA amended with 1.5% (30g/L) chlorate and pairings were carried out on minimal media (MM). Testers from VCGs 0120, 0124, 0125 and 01210 were used to screen the isolate collection. VCG analysis showed that 16 of the isolates belonged to VCG 0124, one of the isolates to VCG 0120 and nine did not form a stable heterokaryon with any of the testers used. Three separate primers sets were used for species and race determination of isolates. A portion of the translation elongation factor 1α (TEF-1α) gene was amplified with primers EF-1/EF-2 and their sequence information used for species identification by comparison to published Fusarium spp. Primer set PBL/PBR, specific for race 1, was used to determine if any of the isolates recovered belonged to this race. Lastly, the specific primers to the tropical race 4 (TR4) strain of FOC, was used to determine if any of the isolates recovered from Puerto Rico belonged to this race. All isolates, including several identified as F. sacchari, caused pathogenic reactions on bananas following inoculation. In addition, pathogenicity tests were useful in determine race structure based on the differential reaction on the set of plants used in inoculations. Twenty two of the isolates were identified as F. oxysporum and six as F. sacchari based on the phenotypic, VCG and molecular tests. Although most of the isolates identified as FOC amplified when using the specific primer set for FOC TR4, the resulting fragment appeared to be larger when compared to control fragment, so the result was associated with a false positive. Based on our results the genetic composition and race structure of the FOC population in Puerto Rico is comprise by race 1 and 2 isolates within the VCG 0124 and it is safe to say that FOC TR4 is absent in the island. The research carried out creates awareness about the plant pathogen, the need to be vigilant in case of outbreaks and suggests that strong quarantine measures are the best approach in keeping out foreign FOC strains.