First Report of Fusarium kalimantanense Causing Fusarium Wilt on Banana Tree in Brazil

Abstract

Banana (Musa spp.) is the second most-consumed fruit in Brazil, the fourth-largest producer globally, with 7 million tons in 2021 (IBGE 2021). Studies about the morphological and pathogenic characteristics revealed that the etiology of Fusarium wilt in banana cultivars in Brazil had been related to the Fusarium oxysporum f. sp. cubense (Foc) (E.F. Smith) Snyder and Hansen species (Costa et al. 2015; Cordeiro et al. 2016; Araújo et al. 2017). Phylogenetic studies have shown the existence of distinct genetic lineages for Foc, which has come to be called the Fusarium oxysporum Species Complex (FOSC) (O'Donnell et al. 1998; Maryani et al. 2019). Symptoms of Fusarium wilt were observed in banana trees at the headquarters of Embrapa Roraima (02°45'26.89"N and 60°43'52.78"W), Roraima-Brazil, in 2016. Samples were collected and sterilized with 70% ethanol for 30 s, followed by 3% NaClO for 1 min, rinsed three times in sterile distilled water, seeded on potato dextrose agar (PDA), and incubated at 25 °C for three days. Two isolates obtained from a pure culture (LPPC130) were submitted to the morphological characterization by Leslie and Summerell (2006) protocol. The fungal colony showed vinaceous color, progressing to livid red (Rayner 1970), with a mean diameter of 41 mm (± 0.1) at three days of incubation in a PDA culture medium. The fungus produced abundant macroconidia in spezieller nährstoffarmer agar (SNA) culture medium containing clove leaf (CLA) after 14 days of incubation at 25 °C. The sporodochium conidia presented a falcate shape, moderately curved, with 3 to 5 septa and dimensions ranging from 38.8 (48.0) 56.2 x 3.5 (4.4) 6.0 µm (n=50). The conidia of the aerial mycelium presented ovoid to ellipsoid shape, slightly curved, aseptic, measuring 6.0 (12.0) 18.0 x 2.8 (3.3) 5.0 µm (n=50). The genomic DNA of the isolate was extracted (Murray and Thompson 1980), and fragments of the elongation factor 1-α (TEF1) and RNA Polymerase II (RPB2) gene regions were amplified and sequenced in both directions (O'Donnell et al. 1998; O'Donnell et al. 2010) (GenBank accession numbers: Seq1 OL802918 and Seq2 OL802919). Multiple alignments of the combined dataset of the isolates and representative sequences obtained from GenBank were submitted to phylogenetic analysis with 1,000 bootstrap replicates. The micromorphological characteristics together to phylogenetic inference on the TEF1 and RPB2 genes, allowed a robust analysis, generating 42 more parsimonious trees and making it possible to identify the LPPC130 isolate as Fusarium kalimantanense, a species belonging to the F. oxysporum species complex (FOSC), with 100% bootstrap support (Maryani et al. 2019). The pathogenicity of the isolate was evaluated in five micropropagated seedlings of banana cv. Silk 75 days old, grown in pots with 5 kg of sterile formulation of sand and soil, in 1:1. Seedlings were inoculated by wounding the roots and depositing a suspension of conidia and chlamydospores at 105 spores mL-1. The inoculating of the isolate in 35 micropropagated seedlings of banana was based on Koch's postulates. The seedlings were transplanted into plastic bags (2 kg of sterile formulation: sandy soil and substrate, in 2:1) and inoculated with 10 mL of the chlamydospore suspension (107 CFU mL-1) at transplanting, and after 30 days of transplanting. Seedlings treated only with water were used as control. Evaluation of the symptoms of the disease was carried out 90 days after inoculation, and revealed that the isolate (LPPC130) was pathogenic by inducing the same symptoms of Fusarium wilt. F. kalimantanense was first reported associated with the pseudostems of Musa acuminata var. Pisang Ambon, and proved to be non-pathogenic to cv. Gros Michel and the bananas of the Cavendish group (Maryani et al. 2019). In Brazil, this fungus was recently associated with the Fusarium rot on melon fruits (Araújo et al. 2021); however, this is the first report of its pathogenicity in banana trees cv. Silk.

Genetic diversity of the banana Fusarium wilt pathogen in Cuba and across Latin America and the Caribbean

“…Recently, the taxonomy of the Fusarium lineages that cause FWB was revised (Maryani et al, 2019). TR4 was considered as the new species F. odoratissimum , Race 2 strains belong to F. phialophorum , F. tardicrescens or F. tardichlamydosporum , while Race 1 strains are present in at least seven species (Czislowski et al, 2021; Maryani et al, 2019; Santos et al, 2022). To determine which lineages or phylogenetic species are present in Cuba and to uncover their genetic diversity, we generated genotyping‐by‐sequencing DArT markers across the 170 Cuban isolates and added these to DArT markers that were previously obtained from 243 additional isolates, comprising 210 isolates from the Americas and the 33 isolates from other regions worldwide (Table S2).…”

“…Phylogenetic analyses, based on several DNA‐based techniques, have consistently shown that Foc isolates are divided into three major clades, which are further subdivided into eight to 10 lineages (Bentley et al, 1998; Fourie et al, 2009; Groenewald et al, 2006; Maryani et al, 2019; Mostert et al, 2017; Mostert et al, 2022; O'Donnell et al, 1998). These lineages encompassing the various physiological races were recently classified as individual phylogenetic species (Maryani et al, 2019), with TR4 strains classified as Fusarium odoratissimum and strains of Race 1 comprising a suite of seven Fusarium spp., while Race 2 strains belong to F. phialophorum, F. tardicrescens or F. tardichlamydosporum (Czislowski et al, 2021; Maryani et al, 2019; Santos et al, 2022). However, this new nomenclature of FWB‐causing Fusarium species generated controversy, due to concerns about the robustness of the classification, which was based on multi‐locus genotyping data (Mostert et al, 2022; Torres Bedoya et al, 2021).…”

“…Clade 3 included a group of 29 F. oxysporum isolates from Brazil, Colombia, Nicaragua, and Peru (Table S2), which did not cluster with any of the representatives of the previously defined lineages. Finally, a small group of isolates formed clade 4, encompassing the F. sangayamense and F. kalimantanense lineages, which are endemic to Indonesia and non-pathogenic on 'Gros Michel' and 'Grand Naine' (Maryani et al, 2019), although the latter was recently reported to infect 'Silk' (AAB) bananas in Brazil, which is indicative for Race 1 (Santos et al, 2022).…”

A deep genetic analysis of banana Fusarium wilt pathogens of Cuba in a Latin American and Caribbean diversity landscape

“…Recently, the taxonomy of the Fusarium lineages that cause FWB was revised (Maryani et al ., 2019). TR4 was considered as the new species F. odoratissimum, Race 2 strains belong to F. phialophorum , F. tardicrescens or F. tardichlamydosporum, while Race 1 strains are present in at least seven species (Maryani et al ., 2019; Czislowski et al ., 2021; Santos et al ., 2022). To determine which species are present in Cuba and to uncover their genetic diversity, we generated genotyping-by-sequencing DArT markers across the 170 Cuban isolates and added these to DArT markers that were previously obtained from 243 additional isolates, comprising 210 isolates from the Americas and the 33 isolates from other regions worldwide (Supporting Information Table S4).…”

“…Clade 3 included a group of 29 F. oxysporum isolates from Brazil, Colombia, Nicaragua, and Perú (Table S3), which did not cluster with any of the representatives of the previously defined lineages. Finally, a small group of isolates formed clade 4, encompassing the F. sangayamense and F. kalimantanense lineages, which are endemic to Indonesia and non-pathogenic on 'Gros Michel' and 'Grand Naine' (Maryani et al, 2019), although the latter was recently reported to infect "Silk" (AAB) bananas in Brazil, which is indicative for Race 1 (Santos et al, 2022). The great majority (95%) of the Cuban isolates grouped in the lineages corresponding with the species F. tardichlamydosporum and F. tardicrescens in clade 2, whereas only 5% grouped in the F. purpurascens lineage of clade 1.…”