MPMI Vol. 36, No. 8, 2023, pp. 529–532, https://doi.org/10.1094/MPMI-10-22-0204-A

Genomic Resources of Four Colletotrichum Species
(C. fioriniae, C. chrysophilum, C. noveboracense,
and C. nupharicola) Threatening Commercial
Apple Production in the Eastern United States

Funding
This material is based on work funded
by the National Institute of Food and
Agriculture through the New York State
Specialty Crop Block Grant Program
2019-2021, project award number
NYFVI 89379/SCG 19 006 to S. G.
Aćimović for the project “qRT-PCR for
Rapid Detection and Differentiation of
Colletotrichum Fungi Causing Fruit
Bitter Rot on New York Apple Farms
and Storages” and from the New York
State Department of Agriculture and
Markets (NYSDAM), Apple Research
and Development Program (ARDP)
project award number NYSDAM
136736 ARDP 6258793 to S. G.
Aćimović for the project titled “Rapid
Molecular Detection and Differentiation
of Colletotrichum Fungi Causing Fruit
Bitter Rot in New York Farms and
Storages by qRT-PCR.”

Keywords
apple bitter rot, Colletotrichum
acutatum species complex, C.
gloeosporioides species complex,
preharvest fruit rot, postharvest fruit rot

Fatemeh Khodadadi,1,2 Emily Giroux,3 Guillaume J. Bilodeau,3 Wayne M. Jurick, II,4

and Srd̄an G. Aćimović2,†

1 University of California Riverside, Department of Plant Pathology and Microbiology, Riverside, CA
92521, U.S.A.

2 Virginia Polytechnic Institute and State University, School of Plant and Environmental Sciences,
Alson H. Smith Jr. Agricultural Research and Extension Center, Winchester, VA 22602, U.S.A.

3 Pathogen Identification Research Laboratory, Ottawa Plant Laboratory, Canadian Food Inspection
Agency, Ottawa, Ontario K2J 4S1, Canada

4 Food Quality Laboratory, U.S. Department of Agriculture, Agriculture Research Service, Beltsville
Agricultural Research Center, Beltsville, MD 20705, U.S.A.

Abstract
The genus Colletotrichum includes nine major clades with 252 species and 15 major phylo-
genetic lineages, also known as species complexes. Colletotrichum spp. are one of the top
fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide.
Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to
98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also
a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable
fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-
Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex
and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex
(CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern
and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but
has been recently found as the second most dominant species causing apple bitter rot
in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581
was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic.
C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter
rot on apple. We deliver the resources of 10 new genomes, including two isolates of C.
fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two
isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra.

The genus Colletotrichum comprises nine major clades containing 252 species and
15 major phylogenetic lineages, also known as species complexes (Cannon et al. 2012;
Talhinhas and Baroncelli 2021). Colletotrichum spp. are one of the top fungal plant
pathogens (Dean et al. 2012), causing anthracnose and pre- and postharvest fruit rot on a
wide range of economically important crops (Cannon et al. 2012). Apple (Malus domestica

†Corresponding author: S. G. Aćimović; acimovic@vt.edu

The author(s) declare no conflict of interest.

Accepted for publication 3 March 2023.

Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND
4.0 International license.

Vol. 36, No. 8, 2023 / 529

https://doi.org/10.1094/MPMI-10-22-0204-A
https://orcid.org/0000-0003-1766-229X
https://orcid.org/0000-0002-0710-2339
mailto:acimovic@vt.edu
https://creativecommons.org/licenses/by-nc-nd/4.0/


Borkh.) is a major agricultural product cultivated in temperate regions worldwide and sig-
nificantly contributes to the human nutrition and global economy. Orchards can suffer dev-
astating losses by bitter rot of apple, which is a serious disease caused by several species
of Colletotrichum (Sutton 2014). Bitter rot was also first described as a major postharvest
pathogen in the Mid-Atlantic region, and it was found that C. fioriniae caused bitter rot in
commercial apple storage facilities (Kou et al. 2014), leading to 2 to 14% unmarketable
fruit. The incidence of apple bitter rot in the Mid-Atlantic United States has risen in the
last 15 years and shifted from minor to highly problematic, with consistent rot problems of
up to 100% loss in organic systems and over 80% in conventional orchards with inade-
quate fungicide spray programs (Khodadadi et al. 2020). Symptoms on apple fruit appear
as small flat and circular spots that turn into large sunken lesions with concentric rings
covered with salmon to black-colored masses of numerous asexual spores.

Dominant species causing apple bitter rot in the Mid-Atlantic are C. fioriniae, from the
Colletotrichum acutatum species complex (CASC), and C. chrysophilum and C. novebo-
racense, from the C. gloeosporioides species complex (CGSC) (Khodadadi et al. 2020;
Martin et al. 2021). C. fioriniae is the dominant species causing apple bitter rot in the
Northeastern and Mid-Atlantic regions of the United States (Khodadadi et al. 2020; Kou
et al. 2014; Martin and Peter 2021; Martin et al. 2021; Munir et al. 2016; Wallhead 2016).
While C. chrysophilum was first identified as an independent novel evolutionary lineage on
banana (Vieira et al. 2017) and cashew (Veloso et al. 2018), in 2020, it was detected as
the second most dominant species causing apple bitter rot in the Mid-Atlantic (Khodadadi
et al. 2020). Using multi-loci phylogenetic analyses, C. noveboracense MB 836581 was
identified as a novel species in the CGSC causing apple bitter rot in the Mid-Atlantic region
(Khodadadi et al. 2020). C. nupharicola is a sister group to C. fructicola and C. noveb-
oracense. C. nupharicola is easily distinguished within the CGSC morphologically and is
reported to cause bitter rot on apple (Leonberger et al. 2019). Species within the CASC and
CGSC differ in crucial characteristics such as lifestyle, temperature optimum, pathogenicity,
and fungicide sensitivity, indicating important differences in their genetic makeup. Correct
species-level detection and identification is critical for developing effective disease control
practices in apple orchards in the United States. In this study, we performed whole-genome
sequencing, de novo assembly, and annotation of two isolates of C. fioriniae (ACFK5,
ACFK16), three isolates of C. chrysophilum (AFK154, AFK26, PMKnsl-1), and two iso-
lates of C. noveboracense (PMBrms-1, AFKH109), collected from bitter rot–infected apple
fruit in New York, Pennsylvania, and Virginia (Khodadadi et al. 2020). The C. novebora-
cense Coll940 isolate was collected from Juglans nigra in Oklahoma, and two isolates of
C. nupharicola (CBS470 and Coll922) were obtained from yellow waterlily (Nuphar lutea)
in Washington and New Jersey, respectively (Doyle et al. 2013).

Total genomic DNA was extracted based on the protocol described by Yelton et al.
(1984), with some modifications. We used fungal mycelia collected from 5-day-old single-
spore cultures grown on potato dextrose agar at room temperature in complete dark-
ness. Except for C. noveboracense isolate AFKH109 (European Nucleotide Archive [ENA]
H1091258; Mycobank as 386581), construction of libraries, whole-genome sequencing,
and quality control were performed by the Beijing Genomics Institute (BGI), using the
DNBSEQ short read platform for 350-bp libraries with the paired-end 150-bp sequenc-
ing strategy, as described in the DNBseq De Novo service overview manual (BGI). Se-
quence adapter trimming and quality control were conducted with Trimmomatic v0.39 and
a phred score cut-off at 20 (Bolger et al. 2014). Genome assemblies were constructed us-
ing SPADES 3.15.2 (isolates Coll940 and AFK154) and megahit 1.2.9 (isolates CBS470,
ACFK5, ACFK16, PMKnsl-1, AFK26, PMBrms-1, and Coll922).

For C. noveboracense AFKH109, the sequencing library was prepared using the Ion
Torrent Ion Xpress Plus fragment library kit (ThermoFisher Scientific) and the Ion Plus
fragment library kit (ThermoFisher Scientific). Genomic DNA was fragmented to 400 bp,
using the Covaris M220 Sonicator (D-Mark Biosciences Inc.). Library fragment size se-
lection was performed using the Pippin Prep (Sage Science) with the 2% DF Marker L
cassette, with the target size set to 300 to 450 bp. Final library amplification was performed
using 12 amplification cycles. A total of 37 pM of sequencing library was loaded onto the
S5 Chef instrument (ThermoFisher Scientific), using the Ion 530 kit (ThermoFisher Sci-
entific), and was sequenced on the Ion Torrent S5 sequencer (ThermoFisher Scientific)

530 / Molecular Plant-Microbe Interactions

https://bgi-com-oss.oss-cn-shenzhen.aliyuncs.com/source/b17ec4bc-2d8d-4035-809b-73d6ddba3465.pdf


Table 1. Colletotrichum species draft genome assembly and completeness summary statistics

C. nupharicola C. fioriniae C. chrysophilum C. noveboracense

Statistics CBS470 Coll922 ACFK16 ACFK5 PMKnsl-1 AFK26 AFK154 Coll940 AFKH109 PMBrms-1

Genome assembly metrics
Genome size (Mb)a 58.75 51.67 49.33 49.44 56.06 55.97 57.85 58.17 58.01 59.08
GC%b 51.03 50.76 52.36 51.94 53.94 52.61 53.21 52.34 52.2 52.59
Coveragec 87× 46× 103× 103× 91× 91× 78× 77× 99× 86×
Contigs 3,696 39,421 514 169 1,954 1,029 1,702 1,007 4,510 2,959
N50 contig length (kb) 137,264 34,056 742,368 688,115 255,107 288,101 268,763 168,995 27,185 176,901
Longest contig length (kb) 938,454 597,000 3,179,133 3,363,293 1,344,106 1,480,977 1,515,687 1,555,225 139,303 1,645,399

Genome annotation metrics
BUSCO completeness (%) 99.2 99 98.8 98.6 98.7 98.9 99.1 99 87.2 99.1
Predicted gene modelsd 13,838 14,983 12,747 12,254 13,816 14,530 14,434 14,420 15,301 13,803
Mean gene length (bp)e 1,510 1,402 1,553 1,542 1,513 1,504 1,485 1,505 1,559 1,517
Genome covered by genes (%) 34.52 25.07 38.97 37.09 36.19 37.97 35.95 36.26 40.06 34.42
Number of genes 13,425 14,546 12,377 11,886 13,409 14,123 13,999 14,011 14,903 13,397

a Genome size calculated from the final processed reads total sequence length.
b GC% calculated from final assembly of processed reads.
c Genome sequencing coverage calculated using the mean read length of the final processed reads (286.74), the calculated genome size, and the number

of read sequences using the coverage/read count calculator (https://stephenturner.shinyapps.io/covcalc/).
d Predicted gene model value includes predicted transfer RNA (tRNA).
e Mean gene length was calculated from coding sequences, excluding predicted tRNA.

(Tremblay and Bilodeau 2022). Sequencing reads were checked for their quality and to
provide graphical guidance for filtering, trimming, and reformatting, using PRINSEQ-LITE
v.0.20.4 and PRINSEQ-graphs v.0.6 software packages (Schmieder and Edwards 2011).
Identification and trimming of sequencing adapters on raw reads were performed using
the AdapterRemoval version 2 package (Schubert et al. 2016). Following adapter removal,
reads were trimmed using a sliding window approach (window size 7, shift 1 base). From
the 3′ end, read bases with the average quality score Q of 7 bases above Q = 10 were
kept. Reads were additionally filtered by complexity score and minimum length (100 bp,
DUST complexity threshold = 7). Genome assembly of the processed reads was per-
formed using Newbler v.2.6 with default parameters (Margulies et al. 2005). Genome an-
notation was performed as described by Giroux and Bilodeau (2020), with modifications of
the reference organisms retrieved from the National Center for Biotechnology Information
used for initial gene predictions within the MAKER pipeline (C. gloeosporioides GSE41844,
Augustus species = Fusarium graminearum, repeat_protein modeling using C. fioriniae
GCA_000582985.1, C. fruticola GCA_000319635.1, C. graminicola GCA_000149035.1,
C. orchidophilum GCA_001831195.1) and when using BUSCO (benchmarking universal
single-copy orthologs) to produce a trained, species-specific hidden Markov model for use
in MAKER (lineage Sordariomyceta).

For all genome annotations reported in this study, the completeness of the genome
assemblies was assessed using BUSCO v5.3.2 with the fungi_odb10 lineage dataset (cre-
ated June 28, 2021) (Simão et al. 2015), and the genome annotation metrics were cal-
culated using AGAT v0.8.1 (Dainat 2022). Summaries of the assembly and annotation
metrics for the species described in this announcement are provided in Table 1. An av-
erage of 16,621 gene models per species assembly were predicted. While the genome
assembly metrics varied for each of the Colletotrichum species sequenced, all genomes
had BUSCO completeness predictions over 95%, except the C. noveboracense AFKH109
genome, which was 87.2%. The GC content of the genomes sequenced ranged from 49.33
to 53.94%, with C. fioriniae and C. chrysophilum having the upper and lower ranges, re-
spectively. The estimated genome sizes ranged from 49.33 to 59.08 Mb. The estimated
sequencing coverage ranged from 46× to 103×, with the lowest and highest coverages
obtained for C. nupharicola and C. fioriniae isolates, respectively.

All strains were deposited to the Agricultural Research Service Culture Collection
(NRRL) at the National Center for Agricultural Utilization Research in Peoria, Illinois,
while those for AFKH109 were deposited in the ENA as H1091258. The genome se-
quences for the above strains have been deposited in GenBank. This work provides new
genomic resources for Colletotrichum spp. that were previously not available and signif-
icantly contributes to the expansion of existing resources for the Colletotrichum genus,
whose species complexes make designing species-specific diagnostic assays challenging.

Vol. 36, No. 8, 2023 / 531

https://stephenturner.shinyapps.io/covcalc/


Thus, the whole-genome sequencing data, obtained from these dominant disease-causing
species, will provide the genome resources needed to identify species-specific gene tar-
gets for molecular marker development to complement existing diagnostic and detection
tools. It is envisioned that these tools will also facilitate many comparative genomics stud-
ies that aim to understand the nature of host-pathogen interactions and factors impacting
disease resistance in the host.

Data Availability
Genome accession numbers in the National Center for Biotechnology Information

GenBank database for the sequenced isolates listed in Table 1 are as follows: CBS470:
GCA_026319135.1, Coll922: GCA_026319225.1, ACFK16: GCA_026319165.1, ACFK5:
GCA_026319145.1, PMKnsl-1: GCA_026319215.1, AFK26: GCA_026319265.1, AFK154:
GCA_026319245.1, Coll940: GCA_026319155.1, AFKH109: GCA_946151405.1,
PMBrms-1: GCA_026319125.1.

Literature Cited
Bolger, A. M., Lohse, M., and Usadel, B. 2014. Trimmomatic: A flexible trimmer for

Illumina Sequence Data. Bioinformatics btu170.
Cannon, P. F., Damm, U., Johnston, P. R., and Weir, B. S. 2012. Colletotrichum -

current status and future directions. Stud. Mycol. 73:181-213.
Dainat, J. 2022. AGAT: Another Gff Analysis Toolkit to handle annotations in any

GTF/GFF format. Version v0.8.1. Zenodo, Geneva.
Dean, R., Van Kan, J. A. L., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro,

A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J., and Foster,
G. D. 2012. The top 10 fungal pathogens in molecular plant pathology. Mol.
Plant Pathol. 13:414-430.

Doyle, V. P., Oudemans, P. V., Rehner, S. A., and Litt, A. 2013. Habitat and host
indicate lineage identity in Colletotrichum gloeosporioides s.I. from wild and
agricultural landscapes in North America. PLoS One 8:e62394.

Giroux, E., and Bilodeau., G. J. 2020. Whole genome sequencing resource of the
European larch canker pathogen Lachnellula willkommii for molecular diagnos-
tic marker development. Phytopathology 110:1255-1259.

Khodadadi, F., González, J. B., Martin, P. L., Giroux, E., Bilodeau, G. J., Peter, K. A.,
Doyle, V. P., and Aćimović, S. G. 2020. Identification and characterization of
Colletotrichum species causing apple bitter rot in New York and description of
C. noveboracense sp. nov. Sci. Rep. 10:11043.

Kou, L., Gaskins, V. L., Luo, Y., and Jurick II, W. M. 2014. First report of Col-
letotrichum fioriniae causing postharvest decay on ‘Nittany’ apple fruit in the
United States. Plant Dis. 98:7.

Leonberger, K., McCulloch, M., and Gauthier, N. W. 2019. Bitter rot of apple. Publi-
cation no. PPFS-FR-T-24. University of Kentucky Cooperative Extension Ser-
vice, Lexington, KY, U.S.A. http://plantpathology.ca.uky.edu/files/ppfs-fr-t-24.
pdf

Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A.,
Berka, J., Braverman, M. S., Chen, Y.-J., and Chen, Z. 2005. Genome sequenc-
ing in microfabricated high-density picolitre reactors. Nature 437:376-380.

Martin, P. L., Krawczyk, T., Khodadadi, F., Aćimović, S. G., and Peter, K. A. 2021.
Bitter rot of apple in the mid-Atlantic United States: Causal species and eval-
uation of the impacts of regional weather patterns and cultivar susceptibility.
Phytopathology 111:966-981.

Martin, P. L., and Peter, K. A. 2021. Quantification of Colletotrichum fioriniae in
orchards and deciduous forests indicates it is primarily a leaf endophyte. Phy-
topathology 111:333-344.

Munir, M., Amsden, B., Dixon, E., Vaillancourt, L., and Gauthier, N. A. W.
2016. Characterization of Colletotrichum species causing bitter rot of apple in
Kentucky orchards. Plant Dis. 100:2194-2203.

Schmieder, R., and Edwards, R. 2011. Quality control and preprocessing of metage-
nomic datasets, Bioinformatics 27:863-864.

Schubert, M., Lindgreen, S., and Orlando, L. 2016. AdapterRemoval v2:
Rapid adapter trimming, identification, and read merging. BMC Res. Notes
9:88.

Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., and Zdobnov,
E. M. 2015. BUSCO: Assessing genome assembly and annotation complete-
ness with single-copy orthologs. Bioinformatics 31:3210-3212.

Sutton, T. B. 2014. Bitter Rot. Pages 20-21 in: Compendium of Apple and Pear
Diseases and Pests. T. B. Sutton, H. S. Aldwinckle, A. M. Agnello, and J. F.
Walgenbach, eds. The American Phytopathological Society, St. Paul, MN,
U.S.A.

Talhinhas, P., and Baroncelli, R. 2021. Colletotrichum species and complexes:
Geographic distribution, host range and conservation status. Fungal Divers.
110:109-198.

Tremblay, E. D., and Bilodeau, G. J. 2022. Biomonitoring of fungal and oomycete
plant pathogens by using metabarcoding. In: Plant Pathology. Methods in
Molecular Biology. Vol. 2536. N. Luchi, ed. Humana, New York.

Veloso, J. S., Câmara, M. P.S., Lima, W. G., Michereff, S. J., and Doyle, V. P. 2018.
Why species delimitation matters for fungal ecology: Colletotrichum diversity on
wild and cultivated cashew in Brazil. Fungal Biol. 122:677-691.

Vieira, W. A. S., Lima, W. G., Nascimento, E. S., Michereff, S. J., Câmara, M. P. S.,
and Doyle, V. P. 2017. The impact of phenotypic and molecular data on the infer-
ence of Colletotrichum diversity associated with Musa. Mycologia 109:912-934.

Wallhead, M. 2016. IPM2.0: Precision agriculture for small-scale crop Pro- duction.
Ph.D. dissertation, University of New Hampshire, Durham, NH, U.S.A.

Yelton, M. M., Hamer, J. E., and Timberlake, W. E. 1984. Transformation of
Aspergillus nidulans by using a trpC plasmid (hybrid plasmid/gene trans-
fer/chromosome integration). Proc. Natl. Acad. Sci. U.S.A. 81:1470-1474.

532 / Molecular Plant-Microbe Interactions

http://plantpathology.ca.uky.edu/files/ppfs-fr-t-24.pdf