Pathogenic, phenotypic and genetic characterization of Fusarium oxysporum f. sp. cubense isolates afeccting banana (Musa spp.) cultivars in Puerto Rico

Abstract

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.

La enfermedad conocida como el mal de Panamá o marchitez por Fusarium, causada por Fusarium oxysporum f. sp. cubense (FOC), es una de las enfermedades más destructivas y ampliamente distribuidas de bananos (Musa spp.). Los objetivos de este estudio fueron: (1) recuperar aislados de FOC de plantas de bananos sintomáticas en Puerto Rico; (2) caracterizar los aislados recuperados patogénica, fenotípica y molecularmente; y (3) determinar los grupos de compatibilidad vegetativa (GCV). En este estudio, se recuperaron 28 aislados monospóricos de plantas sintomáticas de bananos presentes en los municipios de Isabela, Aguada, Mayagüez y Gurabo. Para las pruebas de patogenicidad se utilizaron plantas propagadas mediante cultivo de tejido pertenecientes a los cultivares 2-R-2, 500 (‘Gros Michel’, diferencial para raza 1), ‘Dole’ (‘Bluggoe’, diferencial para raza 2) y ‘5-A’ (‘Cavendish’, diferencial para raza 4). Las inoculaciones se llevaron a cabo utilizando una suspensión de esporas 106 ml-1 . Dos meses luego de la inoculación, se determinó la severidad de la enfermedad tomando en consideración síntomas internos. Los aislados se caracterizaron fenotípicamente basado en la apariencia de la colonia y características morfológicas microscópicas producidas en agar de papa (PDA) y agar de hojas de clavel (CLA). Para las pruebas de complementación de GCV, los mutantes nit se generaron en PDA enmendado con clorato al 1.5% (30g/L) y el apareamiento se llevó a cabo en medio mínimo (MM). Para determinar los GCV que componen la colección de aislados de FOC, se utilizaron los GCV conocidos 0120, 0124, 0125 y 01210. Se amplificó una porción del gen “translation elongation factor 1-α” utilizando los cebadores EF1/EF2 y las secuencias de esta región se utilizó para la identificación a nivel de especie los aislados. Los cebadores PBL/PBR, específicos para la raza 1, se utilizaron para determinar la presencia de esta raza entre nuestros aislados. Por último, los cebadores específicos para la raza 4 tropical (TR4), se utilizaron como método diagnóstico para detectar la presencia de la raza 4 dentro de nuestros aislados. Todos los aislados, incluyendo a los identificados fenotípica y molecularmente como F. sacchari, resultaron patogénicos. Veintidós de los aislados fueron identificados fenotípica y molecularmente como F. oxysporum y seis como F. sacchari. Dieciséis de los aislados formaron un heterocarión robusto con el VCG 0124, mientras que seis de los aislados no formaron un heterocarión con ninguno de los GCV conocidos utilizados. Todos los aislados identificados como FOC amplificaron cuando se utilizaron los cebadores Foc TR4-F/Foc TR4-R, sin embargo el fragmento generado resultó de mayor tamaño que el fragmento esperado, por lo que se consideró un falso positivo. De acuerdo con los resultados obtenidos, la composición genética y estructura de razas de la población de FOC en Puerto Rico está compuesta por aislados pertenecientes a la raza 1 y 2 dentro del VCG 0124. Además se podría concluir que FOC TR4 está ausente en la isla.