GENOME SEQUENCES
Draft Genome Sequence of Fusarium oxysporum f. sp. cubense
Tropical Race 4 from Peru, Obtained by Nanopore and Illumina
Hybrid Assembly
Ana M. Leiva,a Mathieu Rouard,b Diana Lopez-Alvarez,a,c Alberto Cenci,b Catherine Breton,b Rosalyn Acuña,d Juan Carlos Rojas,e
Miguel Dita,f Wilmer J. Cuellara
aCrops for Nutrition and Health, International Center for Tropical Agriculture (CIAT), Palmira, Colombia
bBioversity International, Montpellier, France
cFacultad de Ciencias Agropecuarias, Universidad Nacional de Colombia, Palmira, Colombia
dServicio Nacional de Sanidad Agraria (SENASA), Lima, Peru
eInstituto Nacional de Innovación Agraria (INIA), Lima, Peru
fBioversity International, Cali, Colombia
Ana M. Leiva and Mathieu Rouard contributed equally to this article.
ABSTRACT Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is the causal agent
of Fusarium wilt, a major threat to the banana industry worldwide. Here, we report the
genome of a Foc TR4 strain from Peru, sequenced using a combination of Illumina and
Oxford Nanopore Technologies.
F usarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense, is a devastatingfungal disease affecting bananas worldwide. The pathogen population is divided into
four races, but tropical race 4 (Foc TR4) is by far the most aggressive, as it attacks several
banana types, including Cavendish, which dominates the global banana export economy
(1). In the last 6 years, Foc TR4 has spread from Asia into the Middle East and Africa (2), and
in 2019, Foc TR4 reached Latin America, in the north of Colombia (3).
In April 2021, banana plants (Musa acuminata group AAA, subgroup Cavendish) showing
symptoms of Fusarium wilt were observed on a farm in Querecotillo, Peru (4°43954.840S,
80°33945.000W). Diagnostic analyses confirmed the identity of the pathogen as Foc TR4
(4). Pseudostem strands from symptomatic plants were transferred to potato dextrose
TABLE 1 Representative subset of SIX gene homologs detected in isolates of Fusarium
oxysporum f. sp. cubensea
Presence of SIX gene:
Race VCGb BRIP accession codec 1 2 4 6 7 8 9 13
R1 0123 62895 xd,f xb xb xa xa
R2 01214 25609 xf xa,c xa Editor Antonis Rokas, Vanderbilt University
R4 0122 62892 xc,i xa3 xa xc Copyright © 2022 Leiva et al. This is an open-
STR4 0120 44012 xg xd xa xa xa3,b xa access article distributed under the terms of
TR4 01213 40340 xa,h,i xa xc xa xa1,a2 xa a,e
the Creative Commons Attribution 4.0
x
International license.
TR4d xa,h,i xa xc xa xa1 xa xa,e
Address correspondence to Miguel Dita,
a Isolates shown were reported in reference 12, with the addition of the TR4 Peruvian allelic variants. Sequences m.dita@cgiar.org, or Wilmer J. Cuellar,
were searched on the assembly using BLAST matching with high similarity. X denotes the presence of a gene, w.cuellar@cgiar.org.
while the superscript letters correspond to allelic variants of the gene (TR4 SIX1a, h, i: GenBank accession
The authors declare no conflict of interest.
numbers KX434991, KX434998, KX434999, respectively; SIX2a: KX435000; SIX4c: KX435006; SIX6a: SIX8a1:
KX435011, KX435012; SIX9a: KX435015; SIX13: KX435019, KX435023). SIX, secreted in xylem. Received 11 April 2022
b VCG, vegetative compatibility group. Accepted 12 July 2022
c BRIP, Queensland Plant Pathology Herbarium. Published 8 August 2022
d Peruvian samples.
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FIG 1 Neighbor-joining phylogenetic tree of 17 Fusarium oxysporum f. sp. cubense TR4 isolates with 6,462 SNP variants. Support bootstrap values are indicated as a
percentage of the 1,000 replicates. The sequences included were from Colombia (SRA accession number SRR10747097, SRR10125423, SRR10103605), India (SRR13311628),
Indonesia (SRR10054446), Israel (SRR10054450), Jordan (SRR10054448), Laos (SRR7226878), Lebanon (SRR7226880), Pakistan (SRR7226883), Peru (SRR15514269
to SRR15514272), and the Philippines (SRR10054447) and were mapped onto strain UK0001 (GenBank accession number GCA_007994515.1).
agar (PDA) medium and incubated at 25°C. Single-spore isolates from fungal colonies iden-
tified as Fusarium oxysporum species complex were further purified and used for DNA
extraction (5). DNA from 4 samples (PerS1 to PerS4) was extracted using the Illumina DNA
prep kit and sequenced using the MiSeq platform (2 
 151 bp). The same DNA sample from
PerS4 was further used for sequencing with Oxford Nanopore Technology (FLOW-
MIN111, R10.3 chemistry, LSK109 kit) (6). A total of 46,707,802 Illumina and 379,956 Nanopore
reads (average length, 2,783 bp) were obtained. The filtered reads (Illumina, 93.60% . Q30)
were combined using Unicycler v0.4.8 (7) to make a hybrid genome assembly with a total
length of 46,361,425 bp distributed in 115 contigs (G1C content, 47.59%; N50, 1.63 Mbp). The
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Announcement Microbiology Resource Announcements
consensus sequence quality, checked using Qualimap v2.2.1 (8), resulted in an average depth
of 19.25
 with 6,718,534 reads mapped.
The sequence assembly, mapped using QUAST v5.0.2 (9), showed high contiguity and a
total aligned length of 45.9 Mb (94.7% genome fraction) with the highest-quality genome
sequence available, strain UK0001 (10). Gene space assessment was performed using BUSCO
v5.2.2 (11) (hypocreales odb10), which reported 97.7% completeness (single copy, 97.2%;
duplicate, 0.5%; fragmented, 0.5%; missing, 1.8%; n = 4,494). The hybrid assembly was used to
identify a family of secreted in xylem (SIX) genes. The presence or absence of the SIX homo-
logs was checked using BLASTN v2.2.26 (Table 1) to identify the F. oxysporum f. sp. cubense
sequences and matched the expected allelic variants identified in Foc TR4 for SIX1, SIX6, and
SIX8 (12). Moreover, the PerS4 reads, combined with previously reported Foc TR4 strains (2, 3,
13–15), were mapped on UK0001 (10) using BWA v0.7.15 (16), and single nucleotide polymor-
phism (SNP) calling was conducted using GATK v4.1.6 (17). A dissimilarity matrix (simple
matching index) and a neighbor-joining phylogenetic tree were subsequently computed
using Darwin v6 (18) (Fig. 1). Peruvian samples were clustered together, separated from other
strains, including those from Colombia, suggesting independent incursions of Foc TR4 in the
Americas (Fig. 1). Default parameters were used for all software unless otherwise specified.
The availability of complete genome sequences and their comparative analysis will
contribute to a better understanding of the Foc TR4 population biology, disease epide-
miology, and management.
Data availability. The sequence reads have been deposited at GenBank under the
BioProject accession number PRJNA755905, and the assembly sequence has been de-
posited under the GenBank accession number GCA_021237285.1.
ACKNOWLEDGMENTS
We acknowledge GenLab del Perú S.A.C. for their support with the Illumina sequencing.
We thank Viviana Dominguez from the Cassava Crop Protection Team for technical support.
Special thanks to the Plant Protection team of SENASA Peru. The genomic analysis was
partially realized with the support of MESO@LR and the South Green Platform.
This work was supported in part by grants from STC-CGIAR, Ministerio de Desarrollo
Agrario y Riego (MIDAGRI) from Peru, the CGIAR Research Program on Roots, Tubers and
Bananas (RTB) and CGIAR's Plant Health (PHI) initiative.
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