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Citation: Tan, Q.; Schnabel, G.;
Chaisiri, C.; Yin, L.-F.; Yin, W.-X.;
Luo, C.-X. Colletotrichum Species
Associated with Peaches in China. J.
Fungi 2022,8, 313. https://doi.org/
10.3390/jof8030313
Academic Editors: Samantha C.
Karunarathna, Belle Damodara
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Received: 22 February 2022
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Fungi
Journal of
Article
Colletotrichum Species Associated with Peaches in China
Qin Tan 1, Guido Schnabel 2, Chingchai Chaisiri 1, Liang-Fen Yin 3, Wei-Xiao Yin 3and Chao-Xi Luo 1, 3, *
1Key Lab of Horticultural Plant Biology, Ministry of Education, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China; 15207125880@163.com (Q.T.);
chaisiri.ch@gmail.com (C.C.)
2Department of Plant and Environmental Sciences, Clemson University, Clemson, SC 29634, USA;
schnabe@clemson.edu
3Hubei Key Lab of Plant Pathology, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China; yh@mail.hzau.edu.cn (L.-F.Y.);
wxyin@mail.hzau.edu.cn (W.-X.Y.)
*Correspondence: cxluo@mail.hzau.edu.cn
Abstract:
Colletotrichum is regarded as one of the 10 most important genera of plant pathogens in the
world. It causes diseases in a wide range of economically important plants, including peaches. China
is the largest producer of peaches in the world but little is known about the Colletotrichum spp. affect-
ing the crop. In 2017 and 2018, a total of 286 Colletotrichum isolates were isolated from symptomatic
fruit and leaves in 11 peach production provinces of China. Based on multilocus phylogenetic analy-
ses (ITS, ACT,CAL,CHS-1,GAPDH,TUB2, and HIS3) and morphological characterization, the isolates
were identified to be C. nymphaeae,C. fioriniae, and C. godetiae of the
C. acutatum
species complex,
C. fructicola and C. siamense of the C. gloeosporioides species complex, C. karsti of the
C. boninense
species
complex, and one newly identified species, C. folicola sp. nov. This study is the first report of C. karsti
and C. godetiae in peaches, and the first report of C. nymphaeae,C. fioriniae,C. fructicola, and C. siamense
in peaches in China. C. nymphaeae is the most prevalent species of Colletotrichum in peaches in China,
which may be the result of fungicide selection. Pathogenicity tests revealed that all species found in
this study were pathogenic on both the leaves and fruit of peaches, except for C. folicola, which only
infected the leaves. The present study substantially improves our understanding of the causal agents
of anthracnose on peaches in China.
Keywords: Colletotrichum; peach anthracnose; multilocus phylogeny; pathogenicity; taxonomy
1. Introduction
The peach (Prunus persica (L.) Batsch) originated in China [
1
] and has been grown
in many temperate climates around the world. China is the largest peach producer in
the world, accounting for 55.28% of the total peach acreage in the world and 61.12% of
global peach production [
2
]. The country produced 15,016,103 metric tons on 779,893 ha in
2020 [2].
When the temperature and humidity are favorable, Colletotrichum spp. can infect
peaches and other fruits and cause massive economic losses [
3
]. Colletotrichum spp.
pathogenic on peaches mainly infect the fruit but may also cause leaf or twig lesions.
Fruit lesions appear as firm, brown, sunken (Figure 1a,c,d) areas often displaying con-
centric rings (Figure 1e) of small orange acervuli (Figure 1b,c,f). The acervuli produce
conidia that are primarily spread by rainfall and splashing [
4
]. If a conidium lands on
susceptible host plant tissue, it can cause secondary infection. Gumming can be observed
when Colletotrichum spp. infect fruitlets (Figure 1a). Infected fruitlets do not reach maturity
(Figure 1i), display atrophy, and eventually shrink from water loss (Figure 1i,j). Several
lesions on green or mature fruit may coalesce (Figure 1a,f). Colletotrichum can also infect
leaves with brown lesions (Figure 1g,h) and orange acervuli (Figure 1h). Severe twig
infections can lead to twig dieback (Figure 1j). Colletotrichum species overwinter in fruit
J. Fungi 2022,8, 313. https://doi.org/10.3390/jof8030313 https://www.mdpi.com/journal/jof
J. Fungi 2022,8, 313 2 of 34
mummies and affected twigs, and form conidia in early spring [
5
]. In addition to asexual
reproduction, they may also produce ascospores in perithecia, which were observed on
apples in dead wood and on pears in leaves [6–8].
J. Fungi 2022, 8, x FOR PEER REVIEW 2 of 34
fruit mummies and affected twigs, and form conidia in early spring [5]. In addition to
asexual reproduction, they may also produce ascospores in perithecia, which were ob-
served on apples in dead wood and on pears in leaves [6–8].
a
b c
c
d
e
f g h
i
i
j
j
Figure 1.
Symptoms of peach anthracnose on fruit and leaves. (
a
–
f
) Various symptoms on fruit of
Prunus persica (
a
–
c
,
f
) and P. persica var. nucipersica (
d
,
e
): (
a
,
c
–
e
) lesions on fruitlets and (
b
,
f
) lesions on
mature peach fruit; (
g
,
h
) anthracnose symptoms on leaves; (
i
) mumified young fruit; (
j
) infected twig.
J. Fungi 2022,8, 313 3 of 34
In the past, the taxonomy of the genus Colletotrichum mainly relied on host range
and morphological characteristics [
9
]. However, these characteristics are not suitable
for species-level identification since they are dependent on environmental conditions,
many Colletotrichum species are polyphagous, and multiple species can infect the same
host plant [
10
–
13
]. Molecular identification based on multilocus phylogenetic analyses or
specific gene sequencing has been used for the classification and description of species
concepts [
3
]. To date, 15 Colletotrichum species complexes and 22 individual species have
been identified [14–16].
The causal agents of peach anthracnose were first reported as Colletotrichum acu-
tatum and Colletotrichum gloeosporioides [
17
–
20
]. However, the use of molecular tools
for the classification of anthracnose pathogens revealed that peach anthracnose in the
USA was mostly caused by Colletotrichum nymphaeae and Colletotrichum fioriniae of the
C. acutatum
species complex [
21
], and Colletotrichum siamense and Colletotrichum fructicola
of the
C. gloeosporioides
species complex [
22
]. C. nymphaeae was also reported in Brazil on
peaches [
23
], and C. fioriniae,C. fructicola, and C. siamense were identified in South Korea on
peaches [
24
]. Peach infections by Colletotrichum truncatum and Colletotrichum acutatum are
rare [25,26].
The objective of this study was to systematically identify Colletotrichum spp. associated
with peach fruit and leaf anthracnose in China using morphological characterization and
multilocus phylogenetic analyses.
2. Materials and Methods
2.1. Isolation of Colletotrichum spp. from Peach Samples
During 2017 and 2018, the fruit and leaves of peaches with anthracnose symptoms were
collected from 14 commercial peach orchards and two nurseries (Wuhan, Hubei and Fuzhou,
Fujian) in 11 provinces of China, which were dry-farmed and sprayed with fungicides
for anthracnose control. Conidia on diseased tissues were dipped in a cotton swab and
spread on a potato dextrose agar (PDA, 20% potato infusion, 2% glucose, and
1.5% agar
,
and distilled water) medium and picked up with a glass needle under a professional single
spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China).
If no conidia were present, leaf and fruit pieces (5
×
5 mm) at the intersection of healthy
and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for
30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then
washed three times in sterilized water again. After the tissue pieces were dried, they were
placed on PDA and incubated at 25
◦
C with a 12 h/12 h fluorescent light/dark cycle for
about seven days to produce spores. Cultures were transferred to 15% diluted oatmeal
agar (0.9% oatmeal, 1.5% agar, and distilled water) plates if there was no sporulation on
PDA [
27
]. The ex-type living culture of novel species in this study was deposited in the
China Center for Type Culture Collection (CCTCC), Wuhan, China.
2.2. Morphological Characterization
Mycelial plugs (5 mm) were transferred from the edge of actively growing cultures
to fresh PDA plates and incubated at 25 ◦C in the dark. Colony diameters were measured
after three days to calculate the mycelial growth rates (mm/d). The shape and color of
colonies were investigated on the sixth day. Sexual morphs of some species were produced
after four weeks. The characteristics of conidiomata were observed using fluorescence
stereo microscope (Leica M205 FA, Leica Microsystem Ltd., Wetzlar, Germany). Moreover,
the shape and color of conidia, conidiophores, appressoria, ascomata, asci, ascospores, and
setae were recorded using a light microscope (Nikon Eclipse E400, Nikon Instruments Inc.,
San Francisco, CA, USA), and the length and width of 30 randomly selected conidia and
30 appressoria
were measured for each representative isolate. Appressoria were induced
by dropping 50
µ
L conidial suspension (10
5
conidia/mL) on a microscope slide, which was
placed inside a plate containing moistened filter papers with distilled water, and incubated
at 25 ◦C in the dark for 24 to 48 h [28].
J. Fungi 2022,8, 313 4 of 34
2.3. DNA Extraction, PCR Amplification, and Sequencing
From the 286 obtained isolates, 51 were selected for further multilocus phylogenetic
analyses. They represented each geographical population, colony type, conidia morphology,
and host tissue.
Fungal DNA was extracted as described previously [
29
]. The 5.8S nuclear riboso-
mal gene with the two flanking internal transcribed spacers (ITS), partial sequences of
the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), chitin synthase 1 gene
(
CHS-1
), actin gene (ACT), beta-tubulin gene (TUB2), histone3 gene (HIS3), and calmodulin
gene (CAL) were amplified and sequenced using the primer pairs described in Table S1.
The PCR conditions were 4 min at 95
◦
C, followed by 35 cycles of 95
◦
C for 30 s, annealing
for 30 s at different temperatures for different genes/loci (Table S1), and 72
◦
C for
45 s
, with
a final extension at 72
◦
C for 7 min. DNA sequencing was performed at Tianyi Huiyuan
Biotechnology Co., Ltd. (Wuhan, China) with an ABI 3730XL sequencer from Thermo
Fisher Scientific (China) Co., Ltd. (Shanghai, China). The consensus sequences were as-
sembled from forward and reverse sequences with MEGA v. 7.0 [
30
]. All sequences of
51 representative Colletotrichum isolates in this study were submitted to GenBank and the
accession numbers are listed in Table S2.
2.4. Phylogenetic Analyses
Isolates were divided into four groups based on multilocus phylogenetic analyses,
and type isolates of each species were selected and included in the analyses (Table 1).
Multilocus phylogenetic analyses with concatenated ITS, GAPDH,CHS-1,HIS3,ACT,
and TUB2 sequences were conducted for the C. acutatum species complex [
31
]; ACT,
CAL,CHS-1,GAPDH, ITS, and TUB2 sequences were concatenated for the analysis of
the
C. gloeosporioides
species complex [
32
]; the combined ITS, GAPDH,CHS-1,HIS3,ACT,
TUB2, and CAL sequences were used to analyze the C. boninense species complex [
33
]; and
the ITS, GAPDH,CHS-1,ACT, and TUB2 sequences were applied for remaining species [
34
].
Multiple sequences were aligned and combined using MAFFT v.7 [
35
] and MEGA v.7.0 [
30
].
Bayesian inference (BI) was used to construct phylogenetic trees in MrBayes v.3.2.2 [
36
].
Best-fit models of nucleotide substitution were selected using MrModeltest v.2.3 [
37
] based
on the corrected Akaike information criterion (AIC) (Tables 2–5). BI analyses were launched
with two MCMC chains that were run for 1
×
10
6
generations (C. acutatum species complex
and C. boninense species complex) [
31
,
33
], and trees sampled every 100 generations; or
run 1
×
10
7
generations (C. gloeosporioides species complex, and remaining species) [
8
,
34
],
and trees sampled every 1000 generations. The calculation of BI analyses was stopped
when the average standard deviation of split frequencies fell below 0.01. On this basis, the
first 25% of generations were discarded as burn-in. Maximum parsimony (MP) analyses
were implemented by using Phylogenetic Analysis Using Parsimony (PAUP*) v.4.0b10 [
38
].
Goodness of fit values including tree length (TL), consistency index (CI), retention index
(RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for the
bootstrap analyses (Tables 2–5). Phylogenetic trees were generated using the heuristic
search option with Tree Bisection Reconnection (TBR) branch swapping and 1000 random
sequence additions, with all characters equally weighted and alignment gaps treated as
missing data. Maximum likelihood (ML) analyses were carried out by using the CIPRES
Science Gateway v.3.3 (www.phylo.org, accessed on 29 December 2021), while RAxML-
HPC BlackBox was selected with default parameters. Phylogenetic trees were visualized in
FigTree v.1.4.2 [
39
]. TreeBASE was used to store the concatenated multilocus alignments
(submission number: 29227).
J. Fungi 2022,8, 313 5 of 34
Table 1. Strains used for the phylogenetic analysis of Colletotrichum spp. and other species with details about host, location, and GenBank accession numbers.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
C. acerbum CBS 128530 * Malus domestica New
Zealand
JQ948459 JQ948790 JQ949120 JQ949780 JQ949450 JQ950110 -
C. acutatum CBS 112996 * Carica papaya Australia JQ005776 JQ948677 JQ005797 JQ005839 JQ005818 JQ005860 -
C-1 Prunus persica China KX611163 KY049983 - KY049982 - KY049984 -
C. aenigma ICMP 18608 * Persea americana Israel JX010244 JX010044 JX009774 JX009443 - JX010389 JX009683
C. aeschynomenes ICMP 17673 * Aeschynomene
virginica
USA JX010176 JX009930 JX009799 JX009483 - JX010392 JX009721
C. agaves CBS 118190 Agave striate Mexico DQ286221 - - - - - -
C. alatae ICMP 17919 * Dioscorea alata India JX010190 JX009990 JX009837 JX009471 - JX010383 JX009738
C. alienum ICMP 12071 * Malus domestica New
Zealand
JX010251 JX010028 JX009882 JX009572 - JX010411 JX009654
C. annellatum CBS 129826 * Hevea brasiliensis Colombia JQ005222 JQ005309 JQ005396 JQ005570 JQ005483 JQ005656 JQ005743
C. aotearoa ICMP 18537 * Coprosma sp. New
Zealand
JX010205 JX010005 JX009853 JX009564 - JX010420 JX009611
C. arecicola CGMCC
3.19667 * Areca catechu China MK914635 MK935455 MK935541 MK935374 - MK935498 -
C. artocarpicola MFLUCC
18-1167 *
Artocarpus
heterophyllus
Thailand MN415991 MN435568 MN435569 MN435570 - MN435567 -
C. arxii CBS 132511 * Paphiopedilum sp. Germany KF687716 KF687843 KF687780 KF687802 - KF687881 -
C. asianum ICMP 18580 * Coffea arabica Thailand FJ972612 JX010053 JX009867 JX009584 - JX010406 FJ917506
C. australe CBS 116478 *
Trachycarpus fortunei
South Africa JQ948455 JQ948786 JQ949116 JQ949776 JQ949446 JQ950106 -
C.bambusicola CFCC 54250 * Phyllostachys edulis China MT199632 MT192844 MT192871 MT188638 - MT192817 -
C. beeveri CBS 128527 * Brachyglottis repanda New
Zealand
JQ005171 JQ005258 JQ005345 JQ005519 JQ005432 JQ005605 JQ005692
C. boninense CBS 123755 * Crinum asiaticum
var. sinicum
Japan JQ005153 JQ005240 JQ005327 JQ005501 JQ005414 JQ005588 JQ005674
C. brasiliense CBS 128501 * Passiflora edulis Brazil JQ005235 JQ005322 JQ005409 JQ005583 JQ005496 JQ005669 JQ005756
C. brassicicola CBS 101059 * Brassica oleracea var.
gemmifera
New
Zealand
JQ005172 JQ005259 JQ005346 JQ005520 JQ005433 JQ005606 JQ005693
C. brisbanense CBS 292.67 * Capsicum annuum Australia JQ948291 JQ948621 JQ948952 JQ949612 JQ949282 JQ949942 -
C. cairnsense CBS 140847 * Capsicum annuum Australia KU923672 KU923704 KU923710 KU923716 KU923722 KU923688 -
C. camelliae-japonicae CGMCC
3.18118 *
Camellia japonica Japan KX853165 KX893584 - KX893576 - KX893580 -
J. Fungi 2022,8, 313 6 of 34
Table 1. Cont.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
C. chlorophyti IMI 103806 * Chlorophytum sp. India GU227894 GU228286 GU228384 GU227992 - GU228188 -
C. chrysanthemi IMI 364540 Chrysanthemum
coronarium
China JQ948273 JQ948603 JQ948934 JQ949594 JQ949264 JQ949924 -
C. ciggaro ICMP 18539 * Olea europaea Australia JX010230 JX009966 JX009800 JX009523 - JX010434 JX009635
CBS 237.49 * Hypericum
perforatum
Germany JX010238 JX010042 JX009840 JX009450 - JX010432 JX009636
C. citricola CBS 134228 * Citrus unshiu China KC293576 KC293736 - KC293616 - KC293656 KC293696
C. citrus-medicae HGUP 1554 *,
GUCC 1554
Citrus medica China MN959910 MT006331 MT006328 MT006325 MT006334 - -
GUCC 1555 Citrus medica China MN959911 MT006332 MT006329 MT006326 MT006335 - -
GUCC 1556 Citrus medica China MN959912 MT006333 MT006330 MT006327 MT006336 - -
C. clidemiae ICMP 18658 * Clidemia hirta USA JX010265 JX009989 JX009877 JX009537 - JX010438 JX009645
C. colombiense CBS 129818 * Passiflora edulis Colombia JQ005174 JQ005261 JQ005348 JQ005522 JQ005435 JQ005608 JQ005695
C. constrictum CBS 128504 * Citrus limon New
Zealand
JQ005238 JQ005325 JQ005412 JQ005586 JQ005499 JQ005672 JQ005759
C. cordylinicola ICMP 18579 * Cordyline fruticosa Thailand JX010226 JX009975 JX009864 HM470235 - JX010440 HM470238
C. curcumae IMI 288937 * Curcuma longa India GU227893 GU228285 GU228383 GU227991 - GU228187 -
C. cuscutae IMI 304802 * Cuscuta sp. Dominica JQ948195 JQ948525 JQ948856 JQ949516 JQ949186 JQ949846 -
C. cymbidiicola IMI 347923 * Cymbidium sp. Australia JQ005166 JQ005253 JQ005340 JQ005514 JQ005427 JQ005600 JQ005687
C. dacrycarpi CBS 130241 * Dacrycarpus
dacrydioides
New
Zealand
JQ005236 JQ005323 JQ005410 JQ005584 JQ005497 JQ005670 JQ005757
C. dracaenophilum CBS 118199 * Dracaena sp. China JX519222 JX546707 JX519230 JX519238 - JX519247 -
C. eriobotryae BCRC
FU31138 *
Eriobotrya japonica China MF772487 MF795423 MN191653 MN191648 MN19168 MF795428 -
C. euphorbiae CBS 134725 * Euphorbia sp. South Africa KF777146 KF777131 KF777128 KF777125 KF777247 -
C. fioriniae CBS 128517 * Fiorinia externa USA JQ948292 JQ948622 JQ948953 JQ949613 JQ949283 JQ949943 -
IMI 324996 Malus pumila USA JQ948301 JQ948631 JQ948962 JQ949622 JQ949292 JQ949952 -
CBS 126526 Primula sp. Netherlands JQ948323 JQ948653 JQ948984 JQ949644 JQ949314 JQ949974 -
CBS 124958 Pyrus sp. USA JQ948306 JQ948636 JQ948967 JQ949627 JQ949297 JQ949957 -
CBS 119292 Vaccinium sp. New
Zealand
JQ948313 JQ948643 JQ948974 JQ949634 JQ949304 JQ949964 -
J. Fungi 2022,8, 313 7 of 34
Table 1. Cont.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
ICKb31 Prunus persica South Korea LC516639 LC516653 LC516660 - - LC516646 -
ICKb36 Prunus persica South Korea LC516640 LC516654 LC516661 - - LC516647 -
ICKb47 Prunus persica South Korea LC516641 LC516655 LC516662 - - LC516648 -
C.2.4.2 Prunus persica USA KX066091 KX066094 - - - KX066088 -
CaEY12_1 Prunus persica USA KX066093 KX066096 - - - KX066090 -
C. fructicola ICMP 18581 * Coffea arabica Thailand JX010165 JX010033 JX009866 FJ907426 - JX010405 -
ICMP 18613 Limonium sinuatum Israel JX010167 JX009998 JX009772 JX009491 - JX010388 JX009675
ICMP 18581 * Coffea arabica Thailand JX010165 JX010033 JX009866 FJ907426 - JX010405 FJ917508
ICMP 18727 Fragaria ×ananassa USA JX010179 JX010035 JX009812 JX009565 - JX010394 JX009682
CBS 125397 * Tetragastris
panamensis
Panama JX010173 JX010032 JX009874 JX009581 - JX010409 JX009674
CBS 238.49 * Ficus edulis Germany JX010181 JX009923 JX009839 JX009495 - JX010400 JX009671
ICKb18 Prunus persica South Korea LC516635 LC516649 LC516656 - - LC516642 LC516663
ICKb132 Prunus persica South Korea LC516636 LC516650 LC516657 - - LC516643 LC516664
RR12-3 Prunus persica USA - KJ769247 - - - KM245092 KJ769239
SE12-1 Prunus persica USA - KJ769248 - - - - KJ769237
C. fusiforme MFLUCC 12–
0437 *
unknown Thailand KT290266 KT290255 KT290253 KT290251 - KT290256 -
C. gigasporum CBS 133266 * Centella asiatica Madagascar KF687715 KF687822 KF687761 - - KF687866 -
C. gloeosporioides CBS 112999 * Citrus sinensis Italy JQ005152 JQ005239 JQ005326 JQ005500 JQ005413 JQ005587 JQ005673
ICMP 17821 * Citrus sinensis Italy JX010152 JX010056 JX009818 JX009531 - JX010445 JX009731
C. godetiae CBS 796.72 Aeschynomene
virginica
USA JQ948407 JQ948738 JQ949068 JQ949728 JQ949398 JQ950058 -
CBS 133.44 * Clarkia hybrida Denmark JQ948402 JQ948733 JQ949063 JQ949723 JQ949393 JQ950053 -
IMI 351248 Ceanothus sp. UK JQ948433 JQ948764 JQ949094 JQ949754 JQ949424 JQ950084 -
C. guangxiense CFCC 54251 * Phyllostachys edulis China MT199633 MT192834 MT192861 MT188628 - MT192805 -
C. hippeastri CBS 125376 * Hippeastrum
vittatum
China JQ005231 JQ005318 JQ005405 JQ005579 JQ005492 JQ005665 JQ005752
C. horii ICMP 10492 * Diospyros kaki Japan GQ329690 GQ329681 JX009752 JX009438 JX010450 JX009604
C. indonesiense CBS 127551 * Eucalyptus sp. Indonesia JQ948288 JQ948618 JQ948949 JQ949609 JQ949279 JQ949939 -
C. javanense CBS 144963 * Capsicum annuum Indonesia MH846576 MH846572 MH846573 MH846575 - MH846574 -
J. Fungi 2022,8, 313 8 of 34
Table 1. Cont.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
C. jishouense GZU_HJ2_G2 Nothapodytes
pittosporoides
China MH482931 MH681657 - MH708134 - MH727472 -
C. johnstonii CBS 128532 * Solanum
lycopersicum
New
Zealand
JQ948444 JQ948775 JQ949105 JQ949765 JQ949435 JQ950095 -
C. kahawae IMI 319418 * Coffea arabica Kenya JX010231 JX010012 JX009813 JX009452 - JX010444 -
C. karsti CBS 128524 Citrullus lanatus New
Zealand
JQ005195 JQ005282 JQ005369 JQ005543 JQ005456 JQ005629 JQ005716
CBS 129824 Musa AAA Colombia JQ005215 JQ005302 JQ005389 JQ005563 JQ005476 JQ005649 JQ005736
CBS 128552 Synsepalum
dulcificum
Taiwan JQ005188 JQ005275 JQ005362 JQ005536 JQ005449 JQ005622 JQ005709
C. laticiphilum CBS 112989 * Hevea brasiliensis India JQ948289 JQ948619 JQ948950 JQ949610 JQ949280 JQ949940 -
C. ledebouriae CBS 141284 * Ledebouria floridunda South
Africa
KX228254 - - KX228357 - - -
C. liaoningense CGMCC 3.17616 * Capsicum sp. China KP890104 KP890135 KP890127 KP890097 - KP890111 -
C. limetticola CBS 114.14 * Citrus aurantifolia USA JQ948193 JQ948523 JQ948854 JQ949514 JQ949184 JQ949844 -
C. lindemuthianum CBS 144.31 * Phaseolus vulgaris Germany JQ005779 JX546712 JQ005800 JQ005842 - JQ005863 -
C. magnisporum CBS 398.84 * unknown unknown KF687718 KF687842 KF687782 KF687803 - KF687882 -
C. magnum CBS 519.97 * Citrullus lanatus USA MG600769 MG600829 MG600875 MG600973 - MG601036 -
C. makassarense CBS 143664 * Capsicum annuum Indonesia MH728812 MH728820 MH805850 MH781480 - MH846563 -
C. musae CBS 116870 * Musa sp. USA JX010146 JX010050 JX009896 JX009433 - HQ596280 JX009742
C. neosansevieriae CBS 139918 * Sansevieria trifasciata South
Africa
KR476747 KR476791 - KR476790 - KR476797 -
C. novae-zelandiae CBS 128505 * Capsicum annuum New
Zealand
JQ005228 JQ005315 JQ005402 JQ005576 JQ005489 JQ005662 JQ005749
C. nupharicola ICMP 18187 * Nuphar lutea
subsp.polysepala
USA JX010187 JX009972 JX009835 JX009437 - JX010398 JX009663
C. nymphaeae CBS 515.78 * Nymphaea alba
Netherlands
JQ948197 JQ948527 JQ948858 JQ949518 JQ949188 JQ949848 -
CBS 130.80 Anemone sp. Italy JQ948226 JQ948556 JQ948887 JQ949547 JQ949217 JQ949877 -
IMI 360386 Pelargonium
graveolens
India JQ948206 JQ948536 JQ948867 JQ949527 JQ949197 JQ949857 -
CBS 125973 Fragaria ×ananassa UK JQ948232 JQ948562 JQ948893 JQ949553 JQ949223 JQ949883 -
CaC04_42 Prunus persica USA KX066092 KX066095 - - - KX066089 -
PrpCnSC13–01 Prunus persica Brazil MK761066 MK770424 MK770421 - - MK770427 -
PrpCnSC13–02 Prunus persica Brazil MK765508 MK770425 MK770422 - - MK770428 -
J. Fungi 2022,8, 313 9 of 34
Table 1. Cont.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
PrpCnSC13–10 Prunus persica Brazil MK765507 MK770426 MK770423 - - MK770429 -
C. oncidii CBS 129828 * Oncidium sp. Germany JQ005169 JQ005256 JQ005343 JQ005517 JQ005430 JQ005603 JQ005690
C. orbiculare CBS 570.97 * Cucumis sativus Europe KF178466 KF178490 KF178515 KF178563 - KF178587 -
C. orchidearum CBS 135131 * Dendrobium nobile
Netherlands
MG600738 MG600800 MG600855 MG600944 - MG601005 -
C. orchidophilum CBS 632.80 * Dendrobium sp. USA JQ948151 JQ948481 JQ948812 JQ949472 JQ949142 JQ949802 -
C. parsonsiae CBS 128525 * Parsonsia capsularis New
Zealand
JQ005233 JQ005320 JQ005407 JQ005581 JQ005494 JQ005667 JQ005754
C. paxtonii IMI 165753 * Musa sp.
Saint Lucia
JQ948285 JQ948615 JQ948946 JQ949606 JQ949276 JQ949936 -
C. petchii CBS 378.94 * Dracaena marginata Italy JQ005223 JQ005310 JQ005397 JQ005571 JQ005484 JQ005657 JQ005744
C. phormii CBS 118194 * Phormium sp. Germany JQ948446 JQ948777 JQ949107 JQ949767 JQ949437 JQ950097 -
C. phyllanthi CBS 175.67 * Phyllanthus acidus India JQ005221 JQ005308 JQ005395 JQ005569 JQ005482 JQ005655 JQ005742
C. piperis IMI 71397 * Piper nigrum Malaysia MG600760 MG600820 MG600867 MG600964 - MG601027 -
C. pseudomajus CBS 571.88 * Camellia sinensis China KF687722 KF687826 KF687779 KF687801 - KF687883 -
C. psidii CBS 145.29 * Psidium sp. Italy JX010219 JX009967 JX009901 JX009515 - JX010443 JX009743
C. pyricola CBS 128531 * Pyrus communis New
Zealand
JQ948445 JQ948776 JQ949106 JQ949766 JQ949436 JQ950096 -
C. pyrifoliae CGMCC 3.18902 * Pyrus pyrifolia China MG748078 MG747996 MG747914 MG747768 - MG748158 -
C. queenslandicum ICMP 1778 * Carica papaya Australia JX010276 JX009934 JX009899 JX009447 - JX010414 JX009691
C. radicis CBS 529.93 * unknown Costa Rica KF687719 KF687825 KF687762 KF687785 - KF687869 -
C. salicis CBS 607.94 * Salix sp.
Netherlands
JQ948460 JQ948791 JQ949121 JQ949781 JQ949451 JQ950111 -
C. salsolae ICMP 19051 * Salsola tragus Hungary JX010242 JX009916 JX009863 JX009562 - JX010403 JX009696
C. sansevieriae MAFF 239721 * Sansevieria trifasciata Japan AB212991 - - - - - -
C. scovillei CBS 1265299 * Capsicum sp. Indonesia JQ948267 JQ948597 JQ948928 JQ949588 JQ949258 JQ949918 -
C. siamense ICMP 18578 *,
MFLU 090230
Coffea arabica Thailand JX010171 JX009924 JX009865 FJ907423 - JX010404 FJ917505
C. siamense (syn. C.
hymenocallidis)
CBS 125378 * Hymenocallis
americana
China JX010278 JX010019 GQ856730 GQ856775 - JX010410 JX009709
C. siamense (syn. C.
jasmini-sambac)
CBS 130420 * Jasminum sambac Vietnam HM131511 HM131497 JX009895 HM131507 - JX010415 JX009713
ICKb21 Prunus persica South
Korea LC516637 LC516651 LC516658 - - LC516644 LC516665
ICKb23 Prunus persica South
Korea LC516638 LC516652 LC516659 - - LC516645 LC516666
OD12-1 Prunus persica USA - KJ769240 - - - KM245089 KJ769234
J. Fungi 2022,8, 313 10 of 34
Table 1. Cont.
Species Culture aHost Location GenBank Accession Number
ITS GAPDH CHS-1 ACT HIS3 TUB2 CAL
EY12-1 Prunus persica USA - KJ769246 - - - KM245086 KJ769236
C. simmondsii CBS 122122 * Carica papaya Australia JQ948276 JQ948606 JQ948937 JQ949597 JQ949267 JQ949927 -
C. sloanei IMI 364297 * Theobroma cacao Malaysia JQ948287 JQ948617 JQ948948 JQ949608 JQ949278 JQ949938 -
C. sojae ATCC 62257 * Glycine max USA MG600749 MG600810 MG600860 MG600954 - MG601016 -
C. sydowii CBS 135819 Sambucus sp. China KY263783 KY263785 KY263787 KY263791 - KY263793 -
C. tainanense CBS 143666 * Capsicum annuum Taiwan MH728818 MH728823 MH805845 MH781475 - MH846558 -
C. theobromicola CBS 124945 * Theobroma cacao Panama JX010294 JX010006 JX009869 JX009444 - JX010447 JX009591
C. ti ICMP 4832 * Cordyline sp. New
Zealand
JX010269 JX009952 JX009898 JX009520 - JX010442 JX009649
C. tongrenense GZU_TRJ1-37 Nothapodytes
pittosporoides
China MH482933 MH705332 - MH717074 - MH729805 -
C. torulosum CBS 128544 * Solanum melongena New
Zealand
JQ005164 JQ005251 JQ005338 JQ005512 JQ005425 JQ005598 JQ005685
C. trichellum CBS 217.64 * Hedera helix UK GU227812 GU228204 GU228302 GU227910 - GU228106 -
C. tropicale CBS 124949 * Theobroma cacao Panama JX010264 JX010007 JX009870 JX009489 - JX010407 JX009719
C. truncatum CBS 151.35 * Phaseolus lunatus USA GU227862 GU228254 GU228352 GU227960 - GU228156 -
C. vietnamense CBS 125478 * Coffea sp. Vietnam KF687721 KF687832 KF687769 KF687792 - KF687877 -
C. walleri CBS 125472 * Coffea sp. Vietnam JQ948275 JQ948605 JQ948936 JQ949596 JQ949266 JQ949926 -
C. wanningense CGMCC 3.18936 * Hevea brasiliensis China MG830462 MG830318 MG830302 MG830270 - MG830286 -
C. wuxiense CGMCC 3.17894 * Camellia sinensis China KU251591 KU252045 KU251939 KU251672 - KU252200 KU251833
C. xanthorrhoeae ICMP 17903 * Xanthorrhoea preissii Australia JX010261 JX009927 JX009823 JX009478 - JX010448 JX009653
C. yunnanense CBS 132135 * Buxus sp. China JX546804 JX546706 JX519231 JX519239 - JX519248 -
Monilochaetes infuscans
CBS 869.96 * Ipomoea batatas South
Africa
JQ005780 JX546612 JQ005801 JQ005843 - JQ005864 -
a
CBS: Culture collection of the Centraalbureau voor Schimmelcultures; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; CGMCC: China
General Microbiological Culture Collection; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; IMI: Culture collection of CABI Europe UK Centre, Egham,
UK; BCRC: Bioresource Collection and Research Center, Hsinchu, Taiwan; MFLU: Herbarium of Mae Fah Luang University, Chiang Rai, Thailand; MAFF: MAFF Genebank Project,
Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan; ATCC: American Type Culture Collection. * = Ex-holotype or ex-epitype cultures.
J. Fungi 2022,8, 313 11 of 34
Table 2.
Comparison of alignment properties in parsimony analyses of gene/locus and nucleotide
substitution models used in phylogenetic analyses of C. acutatum species complex.
Gene/Locus ITS GAPDH CHS-1 HIS3 ACT TUB2 Combined
No. of taxa 72 72 68 60 63 72 72
Aligned length
(with gaps) 546 265 282 387 248 492 2240
Invariable characters 501 152 244 289 170 374 1750
Uninformative
variable characters 26 56 13 32 30 60 217
Phylogenetically
informative characters 19 57 25 66 48 58 273
Tree length (TL) 59 176 64 190 117 165 827
Consistency index (CI)
0.85 0.80 0.73 0.66 0.75 0.79 0.71
Retention index (RI) 0.97 0.95 0.94 0.93 0.94 0.94 0.93
Rescaled consistency
index (RC) 0.82 0.76 0.69 0.61 0.71 0.75 0.65
Homoplasy index (HI)
0.15 0.20 0.27 0.34 0.25 0.21 0.30
Nucleotide
substitution model HKY + I HKY + G K80 + I GTR + I + G GTR + G GTR + G GTR + I + G
Table 3.
Comparison of alignment properties in parsimony analyses of gene/locus and nucleotide
substitution models used in phylogenetic analyses of C. gloeosporioides species complex.
Gene/Locus ACT CAL CHS-1 GAPDH ITS TUB2 Combined
No. of taxa 54 58 58 62 58 61 62
Aligned length
(with gaps) 314 744 300 307 614 735 3034
Invariable characters 232 520 239 154 555 489 2209
Uninformative
variable characters 54 139 22 77 36 156 484
Phylogenetically
informative characters 28 85 39 76 23 90 341
Tree length (TL) 115 324 102 264 78 349 1303
Consistency index (CI)
0.84 0.83 0.69 0.75 0.81 0.83 0.76
Retention index (RI) 0.85 0.92 0.84 0.84 0.87 0.87 0.84
Rescaled consistency
index (RC) 0.71 0.76 0.58 0.63 0.70 0.72 0.63
Homoplasy index (HI)
0.17 0.17 0.31 0.25 0.19 0.17 0.24
Nucleotide
substitution model HKY + G GTR + G K80 + G HKY + I SYM + I + G HKY + I GTR + I + G
New species and their most closely related neighbors were analyzed using the Ge-
nealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing
a pairwise homoplasy index (PHI) test [
40
]. The PHI test was carried out on SplitsTree
v.4.14.6 [
41
,
42
] using concatenated sequences (ITS, GAPDH,CHS-1,ACT, and HIS3). The re-
sult of pairwise homoplasy index below a 0.05 threshold (
Φ
w < 0.05) indicated the presence
of significant recombination in the dataset. The relationship between closely related species
was visualized by constructing a splits graph. In addition, the results of relationships
between closely related species were visualized by constructing EqualAngle splits graphs,
using both LogDet character transformation and split decomposition distances options.
J. Fungi 2022,8, 313 12 of 34
Table 4.
Comparison of alignment properties in parsimony analyses of gene/locus and nucleotide
substitution models used in phylogenetic analyses of C. boninense species complex.
Gene/Locus ITS GAPDH CHS-1 HIS3 ACT TUB2 CAL Combined
No. of taxa 25 25 23 23 25 25 24 25
Aligned length
(with gaps) 553 286 280 393 276 502 449 2763
Invariable characters 489 120 224 295 174 348 259 1932
Uninformative variable
characters 40 82 25 28 53 75 103 408
Phylogenetically
informative characters 24 84 31 70 49 79 87 423
Tree length (TL) 87 286 89 210 164 237 300 1404
Consistency index (CI) 0.86 0.80 0.76 0.66 0.82 0.75 0.80 0.76
Retention index (RI) 0.88 0.79 0.79 0.79 0.83 0.75 0.85 0.79
Rescaled consistency
index (RC) 0.75 0.64 0.60 0.52 0.68 0.56 0.70 0.60
Homoplasy index (HI) 0.14 0.20 0.24 0.34 0.18 0.25 0.18 0.24
Nucleotide substitution
model SYM + I + G HKY + I K80 + G GTR + I + G GTR + G HKY + I HKY + G GTR + I + G
Table 5.
Comparison of alignment properties in parsimony analyses of gene/locus and nucleotide
substitution models used in phylogenetic analyses of C. folicola and other taxa.
Gene/Locus ITS GAPDH CHS-1 ACT TUB2 combined
No. of taxa 50 47 44 47 44 50
Aligned length
(with gaps) 571 321 265 279 529 1981
Invariable
characters 367 63 163 102 223 934
Uninformative
variable
characters
53 21 20 39 50 183
Phylogenetically
informative
characters
151 237 82 138 256 864
Tree length (TL)
630 1312 389 671 1300 4405
Consistency
index (CI) 0.51 0.44 0.41 0.48 0.44 0.44
Retention index
(RI) 0.76 0.68 0.66 0.71 0.67 0.68
Rescaled
consistency
index (RC)
0.39 0.30 0.27 0.34 0.30 0.30
Homoplasy
index (HI) 0.49 0.56 0.59 0.53 0.56 0.56
Nucleotide
substitution
model
GTR+I+G HKY+I+G GTR+I+G HKY+I+G HKY+I+G GTR+I+G
2.5. Pathogenicity Test
Two to five isolates of each Colletotrichum sp. were used in pathogenicity tests on
detached fruit and leaves. The experimental varieties for fruit and leaf inoculations were
“Xiaohong” and “Xiahui No. 5”, respectively. Commercially mature fruit (still firm but
with no green background color) and asymptomatic, fully developed leaves with short
twigs (1–2 cm) were washed with soap and water, and surface sterilized in 1% sodium
hypochlorite for 2 min and 30 s, respectively, then rinsed with sterile water and air-dried
on sterile paper. Fruit was stabbed with sterilized toothpicks to produce wounds of about
5 mm deep, while leaves were punctured with sterile, medical needles. For inoculation,
a 10-
µ
L droplet of conidia suspension (1.0
−
2.0
×
10
5
conidia/mL) was dropped on each
J. Fungi 2022,8, 313 13 of 34
wounded site, and control fruit or leaves received sterile water without conidia. Each fruit
and leaf had two inoculation sites. Three fruits and three leaves were used for each isolate.
Inoculated fruit and leaves were placed in a plastic tray onto 30 mm diameter plastic rings
for stability. The bottom of the tray (65 cm
×
40 cm
×
15 cm, 24 peaches or leaves per
tray) contained wet paper towels and the top was sealed with plastic film to maintain
humidity. Peaches and leaves were incubated at 25
◦
C for six days. Pathogenicity was
evaluated by the infection rates and lesion diameters. The infection rates were calculated
by the formula (%) = (infected inoculation sites/all inoculation sites)
×
100%. The lesion
size was determined as the mean of two perpendicular diameters. The experiment was
performed twice.
The fungus was re-isolated from the resulting lesions and identified as described
above, thus fulfilling Koch’s postulates.
3. Results
From 2017 to 2018, a total of 286 Colletotrichum isolates were obtained from 11 provinces
in China (Table 6; Figure 2a); 33 isolates were from leaves and 253 isolates were from fruit
(Table 6). Although we tried to collect samples in Gansu and Shanxi provinces in northern
China, no symptomatic leaves or fruit were found. C. nymphaeae was the most widespread
and most prevalent species (Figure 2b,c), with presence in Hubei, Guizhou, Guangxi, Fujian,
and Sichuan provinces. C. fioriniae was found in three centrally located provinces (Zhejiang,
Guizhou, and Jiangxi). C. siamense was only found in the northernmost orchards of the
collection area in Shandong and Hebei provinces, while C. fructicola was only found in
the southernmost provinces of the collection area of Guangdong and Guizhou provinces.
C. folicola
,C. godetiae, and C. karsti were only found in Yunnan province in the westernmost
border of the collection area (Table 6; Figure 2a).
Table 6.
A list of all Colletotrichum isolates collected from peaches in China based on preliminary
identification.
Species Location Host Number of
Isolates Date Daily Mean
Temperature (◦C) a
C. fioriniae Lishui, Zhejiang Juicy peach,
Yanhong, fruit 17 14 September 2017 29
Tongren, Guizhou Juicy peach, fruit 14 8 August 2018 29
Jian, Jiangxi Yellow peach, fruit 6 21 August 2018 31
C. folicola Honghe, Yunnan Winter peach,
Hongxue, leaf 2 17 August 2017 26
C. fructicola Heyuan,
Guangdong Juicy peach, fruit 19 28 June 2017 29
Shaoguan,
Guangdong
Juicy peach,
Yingzui, fruit 10 3 August 2018 30
Tongren, Guizhou Juicy peach, fruit 10 8 August 2018 29
C. godetiae Honghe, Yunnan Winter peach,
Hongxue, leaf 15 17 August 2017 26
C. karstii Honghe, Yunnan Winter peach,
Hongxue, leaf 3 17 August 2017 26
C. nymphaeae Yichang, Hubei Yellow peach,
NJC83, fruit 11 30 April 2017 19
Jingmen, Hubei Yellow peach,
NJC83, fruit 14 25 April 2017 18
Jingmen, Hubei Juicy peach,
Chunmi, fruit 11 25 April 2017 18
Wuhan, Hubei Juicy peach,
Zaoxianhong, fruit 17 18 April 2017 20
Wuhan, Hubei Flat peach,
Zaoyoupan, fruit 12 18 April 2017 20
J. Fungi 2022,8, 313 14 of 34
Table 6. Cont.
Species Location Host Number of
Isolates Date Daily Mean
Temperature (◦C) a
Wuhan, Hubei Juicy peach, leaft 9 14 June 2017 25
Xiaogan, Hubei Juicy peach,
Chunmei, fruit 4 10 May 2017 20
Qingzhen,
Guizhou
Juicy peach,
Yingqing, fruit 8 21 August 2017 24
Tongren, Guizhou Juicy peach, fruit 2 08 August 2018 29
Guilin, Guangxi Juicy peach,
Chunmi, fruit 38 18 May 2018 25
Guilin, Guangxi Juicy peach,
Chunmi, leaf 4 18 May 2018 25
Fuzhou, Fujian Yellow peach,
huangjinmi, fruit 12 27 July 2018 31
Chengdu, Sichuan
Yellow peach,
Zhongtaojinmi,
fruit
7 28 June 2018 26
C. siamense Qingdao,
Shandong
Juicy peach,
Yangjiaomi, fruit 27 22 August 2017 27
Shijiazhuang,
Hebei
Juicy peach,
Dajiubao, fruit 14 3 August 2018 30
Total 286
aThe average of the daily mean temperatures on the sampling day and the previous six days.
J. Fungi 2022, 8, x FOR PEER REVIEW 16 of 35
Qingzhen, Guizhou
Juicy peach, Yingqing, fruit
8
21 August 2017
24
Tongren, Guizhou
Juicy peach, fruit
2
08 August 2018
29
Guilin, Guangxi
Juicy peach, Chunmi, fruit
38
18 May 2018
25
Guilin, Guangxi
Juicy peach, Chunmi, leaf
4
18 May 2018
25
Fuzhou, Fujian
Yellow peach, huangjinmi, fruit
12
27 July 2018
31
Chengdu, Sichuan
Yellow peach, Zhongtaojinmi, fruit
7
28 June 2018
26
C. siamense
Qingdao, Shandong
Juicy peach, Yangjiaomi, fruit
27
22 August 2017
27
Shijiazhuang, Hebei
Juicy peach, Dajiubao, fruit
14
3 August 2018
30
Total
286
a The average of the daily mean temperatures on the sampling day and the previous six days.
Figure 2. Prevalence of Colletotrichum spp. associated with peaches in China. (a) Map of the
distribution of Colletotrichum spp. on peaches in China. Each color represents one Colletotrichum
species, and the size of the circle indicates the number of isolates collected from that location. (b)
Overall isolation rate (%) of Colletotrichum species; (c) number of sampling locations for each
Colletotrichum species.
3.1. Phylogenetic Analyses
Phylogenetic trees were constructed based on the concatenated gene/locus
sequences. MP and ML trees are not shown because the topologies were similar to the
displayed BI tree (Figure 3, Figure 4, Figure 5 and Figure 6). The number of taxa, aligned
length (with gaps), invariable characters, uninformative variable characters, and
phylogenetically informative characters of each gene/locus and combined sequences are
listed in Table 2, Table 3, Table 4 and Table 5
Figure 2.
Prevalence of Colletotrichum spp. associated with peaches in China. (
a
) Map of the
distribution of Colletotrichum spp. on peaches in China. Each color represents one Colletotrichum
species, and the size of the circle indicates the number of isolates collected from that location.
(
b
) Overall isolation rate (%) of Colletotrichum species; (
c
) number of sampling locations for each
Colletotrichum species.
J. Fungi 2022,8, 313 15 of 34
3.1. Phylogenetic Analyses
Phylogenetic trees were constructed based on the concatenated gene/locus sequences.
MP and ML trees are not shown because the topologies were similar to the displayed BI
tree (Figures 3–6). The number of taxa, aligned length (with gaps), invariable characters,
uninformative variable characters, and phylogenetically informative characters of each
gene/locus and combined sequences are listed in Tables 2–5.
J. Fungi 2022, 8, x FOR PEER REVIEW 17 of 35
For the C. acutatum species complex, in the multilocus sequence analyses
(gene/locus boundaries in the alignment: ITS: 1–546, GAPDH: 551–815, CHS-1: 820–1101,
HIS3: 1106–1492, ACT: 1497–1744, TUB2: 1749–2240) of 27 isolates from peaches in this
study, 44 reference strains of C. acutatum species complex and one Colletotrichum
species (C. orchidophilum strains CBS 632.80) as the outgroup, 2240 characters including
the alignment gaps were processed. For the Bayesian analysis, a HKY + I model was
selected for ITS, a HKY + G model for GAPDH, a K80 + I model for CHS-1, a GTR + I + G
model for HIS3, and a GTR + G model for ACT and TUB2, and all were incorporated in
the analysis (Table 2). As the phylogenetic tree shows in Figure 3, the 27 isolates of the C.
acutatum species complex were clustered in three groups: 11 with C. nymphaeae, eight
with C. fioriniae, and eight with C. godetiae. Although in the same general cluster, C.
nymphaeae from China were genetically distinct from C. nymphaeae isolates from the
USA and Brazil.
Figure 3. A Bayesian inference phylogenetic tree of 71 isolates in the C. acutatum species complex.
C. orchidophilum (CBS 632.80) was used as the outgroup. The tree was built using combined
sequences of the ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2. BI posterior probability values (BI ≥
0.70), MP bootstrap support values (MP ≥ 50%), and RAxML bootstrap support values (ML ≥ 50%)
were shown at the nodes (BI/MP/ML). Tree length = 827, CI = 0.71, RI = 0.93, RC = 0.65, HI = 0.30.
Figure 3.
A Bayesian inference phylogenetic tree of 71 isolates in the C. acutatum species complex.
C. orchidophilum
(CBS 632.80) was used as the outgroup. The tree was built using combined sequences
of the ITS, GAPDH,CHS-1,HIS3,ACT, and TUB2. BI posterior probability values (BI
≥
0.70), MP
bootstrap support values (MP
≥
50%), and RAxML bootstrap support values (ML
≥
50%) were
shown at the nodes (BI/MP/ML). Tree length = 827, CI = 0.71, RI = 0.93, RC = 0.65, HI = 0.30. Ex-type
isolates are in bold. Circles indicate isolates from fruits, and triangles indicate isolates from leaves.
J. Fungi 2022,8, 313 16 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 18 of 35
Ex-type isolates are in bold. Circles indicate isolates from fruits, and triangles indicate isolates from
leaves.
For the C. gloeosporioides species complex, DNA sequences of six genes/loci were
obtained from 19 isolates from peaches in this study, with 42 reference isolates from the
C. gloeosporioides species complex and the outgroup C. boninense CBS 123755. The
gene/locus boundaries of the aligned 3034 characters (with gaps) were: ACT: 1–314, CAL:
319–1062, CHS-1: 1067–1366, GAPDH: 1371–1677, ITS: 1682–2295, TUB2: 2300–3034. For
the Bayesian analysis, a HKY + G model was selected for ACT, a GTR + G model for CAL,
a K80 + G model for CHS-1, a HKY + I model for GAPDH and TUB2, and a SYM + I + G
model for ITS, and they were all incorporated in the analysis (Table 3). In the
phylogenetic tree of the C. gloeosporioides species complex, 10 isolates clustered with C.
fructicola and nine isolates clustered with C. siamense (Figure 4). They clustered together
with isolates from South Korea and the USA.
Figure 4.
A Bayesian inference phylogenetic tree of 61 isolates in the C. gloeosporioides species complex.
C. boninense (CBS 123755) was used as the outgroup. The tree was built using combined sequences
of the ACT,CAL,CHS-1,GAPDH, ITS, and TUB2. BI posterior probability values (BI
≥
0.70), MP
bootstrap support values (MP
≥
50%), and RAxML bootstrap support values (ML
≥
50%) were
shown at the nodes (BI/MP/ML). Tree length = 1303, CI = 0.76, RI = 0.84, RC = 0.63, HI = 0.24. Ex-type
strains are in bold. Circles indicate isolates from fruits, and triangles indicate isolates from leaves.
J. Fungi 2022,8, 313 17 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 19 of 35
Figure 4. A Bayesian inference phylogenetic tree of 61 isolates in the C. gloeosporioides species
complex. C. boninense (CBS 123755) was used as the outgroup. The tree was built using combined
sequences of the ACT, CAL, CHS-1, GAPDH, ITS, and TUB2. BI posterior probability values (BI ≥
0.70), MP bootstrap support values (MP ≥ 50%), and RAxML bootstrap support values (ML ≥ 50%)
were shown at the nodes (BI/MP/ML). Tree length = 1303, CI = 0.76, RI = 0.84, RC = 0.63, HI = 0.24.
Ex-type strains are in bold. Circles indicate isolates from fruits, and triangles indicate isolates from
leaves.
Regarding the C. boninense species complex, in the multilocus analyses (gene/locus
boundaries of ITS: 1–553, GAPDH: 558–843, CHS-1: 848–1127, HIS3: 1132–1524, ACT:
1529–1804, TUB2: 1809–2310, CAL: 2315–2763) of three isolates from peaches in this
study, from 21 reference isolates of C. boninense species complex and one outgroup
strain C. gloeosporioides CBS 112999, 2763 characters including the alignment gaps were
processed. For the Bayesian analysis, a SYM + I + G model was selected for ITS, HKY + I
for GAPDH and TUB2, K80 + G for CHS-1, GTR + I + G for HIS3, GTR + G for ACT, and
HKY + G for CAL, and they were all incorporated in the analysis (Table 4). In Figure 5,
three Chinese isolates clustered with C. karsti in the C. boninense species complex.
Figure 5. A Bayesian inference phylogenetic tree of 24 isolates in the C. boninense species complex.
C. gloeosporioides (CBS 112999) was used as the outgroup. The tree was built using combined
sequences of the ITS, GAPDH, CHS-1, HIS3, ACT, TUB2 and CAL. BI posterior probability values
(BI ≥ 0.70), MP bootstrap support values (MP ≥ 50%), and RAxML bootstrap support values (ML ≥
50%) were shown at the nodes (BI/MP/ML). Tree length = 1404, CI = 0.76, RI = 0.79, RC = 0.60, HI =
0.24. Ex-type strains are in bold. Circles indicate isolates from fruits, and triangles indicate isolates
from leaves.
For the remaining phylogenetic analyses, the alignment of combined DNA
sequences was obtained from 50 taxa, including two isolates from peaches in this study,
Figure 5.
A Bayesian inference phylogenetic tree of 24 isolates in the C. boninense species com-
plex.
C. gloeosporioides
(CBS 112999) was used as the outgroup. The tree was built using combined
sequences of the ITS, GAPDH,CHS-1,HIS3,ACT,TUB2 and CAL. BI posterior probability val-
ues (
BI ≥0.70
), MP bootstrap support values (MP
≥
50%), and RAxML bootstrap support values
(
ML ≥50%
) were shown at the nodes (BI/MP/ML). Tree length = 1404, CI = 0.76, RI = 0.79,
RC = 0.60
,
HI = 0.24. Ex-type strains are in bold. Circles indicate isolates from fruits, and triangles indicate
isolates from leaves.
For the C. acutatum species complex, in the multilocus sequence analyses (gene/locus
boundaries in the alignment: ITS: 1–546, GAPDH: 551–815, CHS-1: 820–1101, HIS3: 1106–1492,
ACT: 1497–1744, TUB2: 1749–2240) of 27 isolates from peaches in this study,
44 reference
strains of C. acutatum species complex and one Colletotrichum species (C. orchidophilum
strains CBS 632.80) as the outgroup, 2240 characters including the alignment gaps were pro-
cessed. For the Bayesian analysis, a HKY + I model was selected for ITS, a
HKY + G model
for GAPDH, a K80 + I model for CHS-1, a
GTR + I + G model
for HIS3, and a
GTR + G model
for ACT and TUB2, and all were incorporated in the analysis (Table 2). As the phylogenetic
tree shows in Figure 3, the 27 isolates of the C. acutatum species complex were clustered
in three groups: 11 with C. nymphaeae, eight with C. fioriniae, and eight with C. godetiae.
Although in the same general cluster, C. nymphaeae from China were genetically distinct
from C. nymphaeae isolates from the USA and Brazil.
J. Fungi 2022,8, 313 18 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 20 of 35
47 reference isolates of Colletotrichum species, and one outgroup strain Monilochaetes
infuscans CBS 869.96. The gene/locus boundaries of the aligned 1981 characters (with
gaps) were: ITS: 1–571, GAPDH: 576–896, CHS-1: 901–1165, ACT: 1170–1448, TUB2:
1453–1981. For the Bayesian analysis, a GTR + I + G model was selected for ITS and
CHS-1, and HKY + I + G for GAPDH, ACT, and TUB2, and they were incorporated in the
analysis (Table 5). In the phylogenetic tree, two isolates (YNHH2-2 and YNHH10-1
(CCTCC M 2020345)) clustered distantly from all known Colletotrichum species and are
described herein as a new species, C. folicola (Figure 6). The PHI test result (Φw = 1) of C.
folicola and its related species C. citrus-medicae ruled out the possibility of gene
recombination interfering with the species delimitation (Figure 7). This is further
evidence that C. folicola is a new species.
Figure 6. A Bayesian inference phylogenetic tree of 49 isolates of Colletotrichum spp. and
outgroup. Monilochaetes infuscans (CBS 869.96) was used as the outgroup. The tree was built
using combined sequences of the ITS, GAPDH, CHS-1, ACT, and TUB2. BI posterior probability
values (BI ≥ 0.70), MP bootstrap support values (MP ≥ 50%), and RAxML bootstrap support values
(ML ≥ 50%) were shown at the nodes (BI/MP/ML). Tree length = 4405, CI = 0.44, RI = 0.68, RC = 0.30,
HI = 0.56. Ex-type strains are in bold. Circles indicate isolates from fruits, and triangles indicate
isolates from leaves.
Figure 6.
A Bayesian inference phylogenetic tree of 49 isolates of Colletotrichum spp. and outgroup.
Monilochaetes infuscans (CBS 869.96) was used as the outgroup. The tree was built using combined
sequences of the ITS, GAPDH,CHS-1,ACT, and TUB2. BI posterior probability values (BI
≥
0.70),
MP bootstrap support values (MP
≥
50%), and RAxML bootstrap support values (ML
≥
50%) were
shown at the nodes (BI/MP/ML). Tree length = 4405, CI = 0.44, RI = 0.68, RC = 0.30, HI = 0.56. Ex-type
strains are in bold. Circles indicate isolates from fruits, and triangles indicate isolates from leaves.
For the C. gloeosporioides species complex, DNA sequences of six genes/loci were
obtained from 19 isolates from peaches in this study, with 42 reference isolates from
the
C. gloeosporioides
species complex and the outgroup C. boninense CBS 123755. The
gene/locus boundaries of the aligned 3034 characters (with gaps) were: ACT: 1–314,
CAL: 319–1062, CHS-1: 1067–1366, GAPDH: 1371–1677, ITS: 1682–2295, TUB2: 2300–3034.
For the Bayesian analysis, a HKY + G model was selected for ACT, a GTR + G model
for CAL, a
K80 + G model
for CHS-1, a HKY + I model for GAPDH and TUB2, and a
SYM + I + G model
for ITS, and they were all incorporated in the analysis (Table 3). In
the phylogenetic tree of the C. gloeosporioides species complex, 10 isolates clustered with
J. Fungi 2022,8, 313 19 of 34
C. fructicola
and nine isolates clustered with C. siamense (Figure 4). They clustered together
with isolates from South Korea and the USA.
Regarding the C. boninense species complex, in the multilocus analyses (gene/locus
boundaries of ITS: 1–553, GAPDH: 558–843, CHS-1: 848–1127, HIS3: 1132–1524, ACT:
1529–1804, TUB2: 1809–2310, CAL: 2315–2763) of three isolates from peaches in this
study, from
21 reference
isolates of C. boninense species complex and one outgroup strain
C. gloeosporioides
CBS 112999, 2763 characters including the alignment gaps were processed.
For the Bayesian analysis, a SYM + I + G model was selected for ITS, HKY + I for GAPDH
and TUB2, K80 + G for CHS-1, GTR + I + G for HIS3, GTR + G for ACT, and HKY + G for
CAL, and they were all incorporated in the analysis (Table 4). In Figure 5, three Chinese
isolates clustered with C. karsti in the C. boninense species complex.
For the remaining phylogenetic analyses, the alignment of combined DNA sequences
was obtained from 50 taxa, including two isolates from peaches in this study, 47 reference
isolates of Colletotrichum species, and one outgroup strain Monilochaetes infuscans CBS
869.96. The gene/locus boundaries of the aligned 1981 characters (with gaps) were: ITS:
1–571, GAPDH: 576–896, CHS-1: 901–1165, ACT: 1170–1448, TUB2: 1453–1981. For the
Bayesian analysis, a GTR + I + G model was selected for ITS and CHS-1, and HKY + I
+ G for GAPDH,ACT, and TUB2, and they were incorporated in the analysis (Table 5).
In the phylogenetic tree, two isolates (YNHH2-2 and YNHH10-1 (CCTCC M 2020345))
clustered distantly from all known Colletotrichum species and are described herein as a new
species,
C. folicola
(Figure 6). The PHI test result (
Φ
w = 1) of C. folicola and its related species
C. citrus-medicae ruled out the possibility of gene recombination interfering with the species
delimitation (Figure 7). This is further evidence that C. folicola is a new species.
J. Fungi 2022, 8, x FOR PEER REVIEW 21 of 35
Figure 7. PHI test of C. folicola and phylogenetically related species using both LogDet
transformation and splits decomposition. PHI test value (Φw) < 0.05 indicate significant
recombination within the datasets.
3.2. Taxonomy
Colletotrichum nymphaeae H.A. van der Aa, Netherlands Journal of Plant
Pathology. 84: 110. (1978) (Figure 8).
Description and illustration—Damm et al. [31].
Figure 8. Biological characteristics of Colletotrichum nymphaeae. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores ((a–e)
isolate HBYC 1; (f) isolate SCCD 1). Scale bars: (c) = 200 μm; (d–f) = 20 μm.
Materials examined: China, Hubei province, Yichang city, on fruit of P. persica cv.
NJC83, April 2017, Q. Tan, living culture HBYC1; Sichuan province, Chengdu city, on
Figure 7.
PHI test of C. folicola and phylogenetically related species using both LogDet transformation
and splits decomposition. PHI test value (
Φ
w) < 0.05 indicate significant recombination within
the datasets.
3.2. Taxonomy
Colletotrichum nymphaeae H.A. van der Aa, Netherlands Journal of Plant Pathology.
84: 110
.
(1978) (Figure 8).
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J. Fungi 2022, 8, x FOR PEER REVIEW 20 of 34
Figure 7. PHI test of C. folicola and phylogenetically related species using both LogDet transfor-
mation and splits decomposition. PHI test value (Φw) < 0.05 indicate significant recombination
within the datasets.
3.2. Taxonomy
Colletotrichum nymphaeae H.A. van der Aa, Netherlands Journal of Plant Pathology. 84:
110. (1978) (Figure 8).
Description and illustration—Damm et al. [31].
Figure 8. Biological characteristics of Colletotrichum nymphaeae. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores ((a–e) iso-
late HBYC 1; (f) isolate SCCD 1). Scale bars: (c) = 200 μm; (d–f) = 20 μm.
Materials examined: China, Hubei province, Yichang city, on fruit of P. persica cv.
NJC83, April 2017, Q. Tan, living culture HBYC1; Sichuan province, Chengdu city, on
Figure 8.
Biological characteristics of Colletotrichum nymphaeae. (
a
,
b
) Front and back view of six-day-
old PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
) appressoria; (
f
) conidiophores ((
a
–
e
) isolate HBYC
1; (f) isolate SCCD 1). Scale bars: (c) = 200 µm; (d–f) = 20 µm.
Description and illustration—Damm et al. [31].
Materials examined: China, Hubei province, Yichang city, on fruit of P. persica cv.
NJC83, April 2017, Q. Tan, living culture HBYC1; Sichuan province, Chengdu city, on fruit
of P. persica cv. Zhongtaojinmi, June 2018, Q. Tan, living culture SCCD 1; Fujian province,
Fuzhou city, on fruit of P. persica cv. Huangjinmi, July 2018, Q. Tan, living culture FJFZ 1;
Guangxi province, Guilin city, on leaves of P. persica cv. Chunmei, May 2018, Q. Tan, living
culture GXGL 13-1; Guizhou province, Tongren city, on fruit of P. persica, June 2018, Q.
Tan living culture GZTR 8-1; Hubei province, Jingmen city, on fruit of P. persica cv. NJC83,
April 2018
, Q. Tan, living culture HBJM 1-1; Hubei province, Wuhan city, on fruit of P.
persica var. nucipersica cv. Zhongtaojinmi, April 2017, Q. Tan, living culture HBWH 2-1;
ibid, on leaves of P. persica, June 2017, L.F. Yin, living culture HBWH 3-2; Hubei province,
Xiaogan city, on fruit of P. persica cv. Chunmei, May 2017, Q. Tan, living culture HBXG 1.
Notes: Colletotrichum nymphaeae was first described on leaves of Nymphaea alba in Ko-
rtenhoef by Van der Aa [
43
]. C. nymphaeae is well separated from other species with
TUB2, but all other genes have very high intraspecific variability [
31
]. Consistently,
C. nymphaeae isolates collected in this study are different from ex-type strain CBS 515.78 in
ITS (2 bp), GAPDH (1 bp), CHS-1 (3 bp), ACT (1 bp), HIS3 (3 bp), but with 100% identity
in TUB2.
Colletotrichum fioriniae (Marcelino and Gouli) R.G. Shivas and Y.P. Tan, Fungal Diversity
39: 117. (2009) (Figure 9).
J. Fungi 2022,8, 313 21 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 21 of 34
fruit of P. persica cv. Zhongtaojinmi, June 2018, Q. Tan, living culture SCCD 1; Fujian
province, Fuzhou city, on fruit of P. persica cv. Huangjinmi, July 2018, Q. Tan, living
culture FJFZ 1; Guangxi province, Guilin city, on leaves of P. persica cv. Chunmei, May
2018, Q. Tan, living culture GXGL 13-1; Guizhou province, Tongren city, on fruit of P.
persica, June 2018, Q. Tan living culture GZTR 8-1; Hubei province, Jingmen city, on fruit
of P. persica cv. NJC83, April 2018, Q. Tan, living culture HBJM 1-1; Hubei province,
Wuhan city, on fruit of P. persica var. nucipersica cv. Zhongtaojinmi, April 2017, Q. Tan,
living culture HBWH 2-1; ibid, on leaves of P. persica, June 2017, L.F. Yin, living culture
HBWH 3-2; Hubei province, Xiaogan city, on fruit of P. persica cv. Chunmei, May 2017, Q.
Tan, living culture HBXG 1.
Notes: Colletotrichum nymphaeae was first described on leaves of Nymphaea alba in
Kortenhoef by Van der Aa [43]. C. nymphaeae is well separated from other species with
TUB2, but all other genes have very high intraspecific variability [31]. Consistently, C.
nymphaeae isolates collected in this study are different from ex-type strain CBS 515.78 in
ITS (2 bp), GAPDH (1 bp), CHS-1 (3 bp), ACT (1 bp), HIS3 (3 bp), but with 100% identity
in TUB2.
Colletotrichum fioriniae (Marcelino and Gouli) R.G. Shivas and Y.P. Tan, Fungal Di-
versity 39: 117. (2009) (Figure 9).
Description and illustration—Damm et al. [31].
Figure 9. Biological characteristics of Colletotrichum fioriniae. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores ((a–e) iso-
late JXJA 6; (f) isolate JXJA 1). Scale bars: (c) = 200 μm; (d–f) = 20 μm.
Materials examined: China, Jiangxi province, Jian city, on fruit of P. persica, August
2018, Q. Tan, living cultures JXJA 1, JXJA 6; Zhejiang province, Lishui city, on fruit of P.
persica, September 2017, Q. Tan, living cultures ZJLS 1, ZJLS 11-1; Guizhou province,
Tongren city, on fruit of P. persica, August 2018, Q. Tan, living culture GZTR 7-1.
Notes: Colletotrichum acutatum var. fioriniae was first isolated from Fiorinia externa
[44] and host plants of the scale insect as an endophyte [45] in New York, USA. In 2009,
Shivas and Tan identified it from Acacia acuminate, Persea americana, and Mangifera indica
Figure 9.
Biological characteristics of Colletotrichum fioriniae. (
a
,
b
) Front and back view of six-day-old
PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
) appressoria; (
f
) conidiophores ((
a
–
e
) isolate JXJA 6;
(f) isolate JXJA 1). Scale bars: (c) = 200 µm; (d–f) = 20 µm.
Description and illustration—Damm et al. [31].
Materials examined: China, Jiangxi province, Jian city, on fruit of P. persica, August
2018, Q. Tan, living cultures JXJA 1, JXJA 6; Zhejiang province, Lishui city, on fruit of
P. persica, September 2017, Q. Tan, living cultures ZJLS 1, ZJLS 11-1; Guizhou province,
Tongren city, on fruit of P. persica, August 2018, Q. Tan, living culture GZTR 7-1.
Notes: Colletotrichum acutatum var. fioriniae was first isolated from Fiorinia externa [
44
]
and host plants of the scale insect as an endophyte [
45
] in New York, USA. In 2009,
Shivas and Tan identified it from Acacia acuminate,Persea americana, and Mangifera indica
in Australia as a separate species and named it Colletotrichum fioriniae [46]. C. fioriniae was
mainly isolated from wide host plants and fruits in the temperate zones [
3
,
31
]. In this study,
the C. fioriniae isolates clustered in two subclades, which is consistent with the results of
Damm’s study [31].
Colletotrichum godetiae P. Neergaard, Friesia 4: 72. (1950) (Figure 10).
Description and illustration—Damm et al. [31].
Materials examined: China, Yunnan Province, Honghe City, on leaves of P. persica cv.
Hongxue, August 2017, Q. Tan, living cultures YNHH 1-1, YNHH 4-1, YNHH 6-1, YNHH
8-2 and YNHH 9-1.
Notes: Colletotrichum godetiae was first reported on the seeds of Godetia hybrid in
Denmark by Neergaard in 1943 [
47
], and given detailed identification seven years later [
48
].
C. godetiae was also recovered from fruits of Fragaria
×
ananassa,Prunus cerasus,Solanum
betaceum,Citrus aurantium, and Olea europaea [
49
]; leaves of Laurus nobilis and Mahonia
aquifolium; twigs of Ugni molinae; and canes of Rubus idaeus [
31
]. In this study, the isolates
were obtained from peach leaves and could infect both the peach fruit and leaf.
J. Fungi 2022,8, 313 22 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 22 of 34
in Australia as a separate species and named it Colletotrichum fioriniae [46]. C. fioriniae was
mainly isolated from wide host plants and fruits in the temperate zones [3,31]. In this
study, the C. fioriniae isolates clustered in two subclades, which is consistent with the
results of Damm’s study [31].
Colletotrichum godetiae P. Neergaard, Friesia 4: 72. (1950) (Figure 10).
Description and illustration—Damm et al. [31].
Figure 10. Biological characteristics of Colletotrichum godetiae. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e–h) appressoria; (i) conidiophores ((a–f,i)
isolate YNHH 1-1, (g,h) YNHH 9-1). Scale bars: (c) = 200 μm; (d–i) = 20 μm.
Materials examined: China, Yunnan Province, Honghe City, on leaves of P. persica
cv. Hongxue, August 2017, Q. Tan, living cultures YNHH 1-1, YNHH 4-1, YNHH 6-1,
YNHH 8-2 and YNHH 9-1.
Notes: Colletotrichum godetiae was first reported on the seeds of Godetia hybrid in
Denmark by Neergaard in 1943 [47], and given detailed identification seven years later
[48]. C. godetiae was also recovered from fruits of Fragaria × ananassa, Prunus cerasus, So-
lanum betaceum, Citrus aurantium, and Olea europaea [49]; leaves of Laurus nobilis and Ma-
Figure 10.
Biological characteristics of Colletotrichum godetiae. (
a
,
b
) Front and back view of six-day-old
PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
–
h
) appressoria; (
i
) conidiophores ((
a
–
f
,
i
) isolate YNHH
1-1, (g,h) YNHH 9-1). Scale bars: (c) = 200 µm; (d–i) = 20 µm.
Colletotrichum fructicola H. Prihastuti et al., Fungal Diversity 39: 96. (2009) (Figure 11).
Description and illustration—Prihastuti et al. [50].
Materials examined: China, Guangdong province, Heyuan city, on fruit of P. persica,
June 2017, Q. Tan, living culture GDHY 10-1; Guangdong province, Shaoguan city, on fruit
of P. persica cv. Yingzuitao, August 2018, Q. Tan, living cultures GDSG 1-1, GDSG 5-1;
Guizhou province, Tongren city, on fruit of P. persica, August 2018, Q. Tan, living culture
GZTR 10-1.
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honia aquifolium; twigs of Ugni molinae; and canes of Rubus idaeus [31]. In this study, the
isolates were obtained from peach leaves and could infect both the peach fruit and leaf.
Colletotrichum fructicola H. Prihastuti et al., Fungal Diversity 39: 96. (2009) (Figure 11).
Description and illustration—Prihastuti et al. [50].
Figure 11. Biological characteristics of Colletotrichum fructicola. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores; (g) asco-
mata; (h,i) asci; (j) ascospores ((a–e) isolate GDHY 10-1; (f–j) isolate GDSG 1-1). Scale bars: (c) = 200
μm; (d–j) = 20 μm.
Materials examined: China, Guangdong province, Heyuan city, on fruit of P. persica,
June 2017, Q. Tan, living culture GDHY 10-1; Guangdong province, Shaoguan city, on
fruit of P. persica cv. Yingzuitao, August 2018, Q. Tan, living cultures GDSG 1-1, GDSG
5-1; Guizhou province, Tongren city, on fruit of P. persica, August 2018, Q. Tan, living
culture GZTR 10-1.
Notes: Colletotrichum fructicola was first described from the berries of Coffea arabica in
Chiang Mai Province, Thailand [50]. Subsequently, C. fructicola was reported on a wide
range of hosts including Malus domestica, Fragaria × ananassa, Limonium sinuatum, Pyrus
pyrifolia, Dioscorea alata, Theobroma cacao Vaccinium spp., Vitis vinifera, and Prunus persica
Figure 11.
Biological characteristics of Colletotrichum fructicola. (
a
,
b
) Front and back view of six-
day-old PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
) appressoria; (
f
) conidiophores; (
g
) ascomata;
(
h
,
i
) asci; (
j
) ascospores ((
a
–
e
) isolate GDHY 10-1; (
f
–
j
) isolate GDSG 1-1). Scale bars: (
c
) = 200
µ
m;
(d–j) = 20 µm.
Notes: Colletotrichum fructicola was first described from the berries of Coffea ara-
bica in Chiang Mai Province, Thailand [
50
]. Subsequently, C. fructicola was reported
on a wide range of hosts including Malus domestica,Fragaria
×
ananassa,Limonium sin-
uatum,Pyrus pyrifolia,Dioscorea alata,Theobroma cacao Vaccinium spp., Vitis vinifera, and
Prunus persica [3,51]
. In this study, the conidia and ascospores of C. fructicola isolates
(
9.3−18.9 ×3.4−8.2 µm
,
mean ±SD = 14.3 ±1.7 ×5.6 ±0.5 µm
; 12.6
−
22.0
×
3.1–7.6
µ
m,
mean ±SD = 17.3 ±0.5 ×5.0 ±0.5 µm) (Table S3)
were larger than that of ex-type (MFLU
090228, ICMP 185819: 9.7
−
14
×
3
−
4.3
µ
m, mean
±
SD = 11.53
±
1.03
×
3.55
±
0.32
µ
m;
9−14 ×3–4 µm, mean ±SD = 11.91 ±1.38 ×3.32 ±0.35 µm).
J. Fungi 2022,8, 313 24 of 34
Colletotrichum siamense H. Prihastuti et al., Fungal Diversity 39: 98. (2009) (Figure 12).
J. Fungi 2022, 8, x FOR PEER REVIEW 24 of 34
[3,51]. In this study, the conidia and ascospores of C. fructicola isolates (9.3−18.9 × 3.4−8.2
μm, mean ± SD = 14.3 ± 1.7 × 5.6 ± 0.5 μm; 12.6−22.0 × 3.1–7.6 μm, mean ± SD = 17.3 ± 0.5 ×
5.0 ± 0.5 μm) (Table S3) were larger than that of ex-type (MFLU 090228, ICMP 185819:
9.7−14 × 3−4.3 μm, mean ± SD = 11.53 ± 1.03 × 3.55 ± 0.32 μm; 9−14 × 3–4 μm, mean ± SD =
11.91 ± 1.38 × 3.32 ± 0.35 μm).
Colletotrichum siamense H. Prihastuti et al., Fungal Diversity 39: 98. (2009) (Figure 12).
Description and illustration—Prihastuti et al. [50].
Figure 12. Biological characteristics of Colletotrichum siamense. (a,b) Front and back view of
six-day-old PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores ((a–e) iso-
late SDQD10-1; (f) isolate HBSJZ 1-1). Scale bars: (c) = 200 μm; (d–f) = 20 μm.
Materials examined: China, Shandong province, Qingdao city, on fruit of P. persica
cv. Yangjiaomi, August 2017, Q. Tan, living cultures SDQD 1-1, SDQD 10-1; Hebei
province, Shijiazhuang city, on fruit of P. persica cv. Dajiubao, August 2018, Q. Tan, living
cultures HBSJZ 1-1, HBSJZ 3-1.
Notes: Colletotrichum siamense was first identified on the berries of Coffea arabica in
Chiang Mai Province, Thailand [50] and reported to have a wide range of hosts across
several tropical, subtropical, and temperate regions, including Persea americana and Carica
papaya in South Africa; Fragaria × ananassa, Vitis vinifera, and Malus domestica in the USA;
Hymenocallis americana and Pyrus pyrifolia in China; etc. [3,8,51]. In this study, we collected
C. siamense isolates from the temperate zone in China; the conidia (13.2−18.3 × 4.6–6.3 μm,
mean ± SD = 15.3 ± 0.4 × 5.4 ± 0.3 μm) (Table S3) were larger than those of the ex-holotype
(MFLU 090230, ICMP 18578: 7–18.3 × 3–4.3 μm, mean ± SD = 10.18 ± 1.74 × 3.46 ± 0.36 μm).
Colletotrichum karsti Y.L. Yang et al., Cryptogamie Mycologie. 32: 241. (2011) (Figure
13).
Description and illustration—Yang et al. [52].
Figure 12.
Biological characteristics of Colletotrichum siamense. (
a
,
b
) Front and back view of six-
day-old PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
) appressoria; (
f
) conidiophores ((
a
–
e
) isolate
SDQD10-1; (f) isolate HBSJZ 1-1). Scale bars: (c) = 200 µm; (d–f) = 20 µm.
Description and illustration—Prihastuti et al. [50].
Materials examined: China, Shandong province, Qingdao city, on fruit of P. persica cv.
Yangjiaomi, August 2017, Q. Tan, living cultures SDQD 1-1, SDQD 10-1; Hebei province,
Shijiazhuang city, on fruit of P. persica cv. Dajiubao, August 2018, Q. Tan, living cultures
HBSJZ 1-1, HBSJZ 3-1.
Notes: Colletotrichum siamense was first identified on the berries of Coffea arabica in
Chiang Mai Province, Thailand [
50
] and reported to have a wide range of hosts across
several tropical, subtropical, and temperate regions, including Persea americana and Carica
papaya in South Africa; Fragaria
×
ananassa,Vitis vinifera, and Malus domestica in the USA;
Hymenocallis americana and Pyrus pyrifolia in China; etc. [
3
,
8
,
51
]. In this study, we collected
C. siamense isolates from the temperate zone in China; the conidia (
13.2−18.3 ×4.6–6.3 µm
,
mean ±SD = 15.3 ±0.4 ×5.4 ±0.3 µm
) (Table S3) were larger than those of the ex-holotype
(MFLU 090230, ICMP 18578: 7–18.3
×
3–4.3
µ
m,
mean ±SD = 10.18 ±1.74 ×3.46 ±0.36 µm
).
Colletotrichum karsti Y.L. Yang et al., Cryptogamie Mycologie. 32: 241. (2011) (Figure 13).
Description and illustration—Yang et al. [52].
Materials examined: China, Yunnan province, Honghe city, on leaves of P. persica cv.
Hongxue, August 2017, Q. Tan, living cultures YNHH 3-1, YNHH 3-2, and YNHH 5-2.
Notes: Colletotrichum karsti was first described from Vanda sp. (Orchidaceae) as a
pathogen on diseased leaf and endophyte of roots in Guizhou province, China [
52
].
C. karsti
is the most common and geographically diverse species in the C. boninense species complex,
and occurs on wild hosts including Vitis vinifera,Capsicum spp., Lycopersicon esculentum,
Coffea sp., Citrus spp., Musa banksia,Passiflora edulis,Solanum betaceum,Zamia obliqua,
J. Fungi 2022,8, 313 25 of 34
etc. [
11
,
33
,
52
,
53
]. In this study, the conidia of C. karsti isolates (
10.6 −14.9 ×5.8−7.4 µm
,
mean
±
SD = 12.9
±
0.3
×
6.7
±
0.2
µ
m) (Table S3) were smaller than those of the ex-
holotype (CGMCC3.14194: 12–19.5 ×5–7.5 µm, mean ±SD = 15.4 ±1.3 ×6.5 ±0.5 µm).
J. Fungi 2022, 8, x FOR PEER REVIEW 25 of 34
Figure 13. Biological characteristics of Colletotrichum karsti. (a,b) Front and back view of six-day-old
PDA culture; (c) conidiomata; (d) conidia; (e) appressoria; (f) conidiophores; (g) ascomata; (h,i) as-
ci; (j) ascospores ((a–j) isolate YNHH 3-1). Scale bars: (c) = 200 μm; (d–j) = 20 μm.
Materials examined: China, Yunnan province, Honghe city, on leaves of P. persica cv.
Hongxue, August 2017, Q. Tan, living cultures YNHH 3-1, YNHH 3-2, and YNHH 5-2.
Notes: Colletotrichum karsti was first described from Vanda sp. (Orchidaceae) as a
pathogen on diseased leaf and endophyte of roots in Guizhou province, China [52]. C.
karsti is the most common and geographically diverse species in the C. boninense species
complex, and occurs on wild hosts including Vitis vinifera, Capsicum spp., Lycopersicon
esculentum, Coffea sp., Citrus spp., Musa banksia, Passiflora edulis, Solanum betaceum, Zamia
obliqua, etc. [11,33,52,53]. In this study, the conidia of C. karsti isolates (10.6 − 14.9 × 5.8−7.4
μm, mean ± SD = 12.9 ± 0.3 × 6.7 ± 0.2 μm) (Table S3) were smaller than those of the
ex-holotype (CGMCC3.14194: 12–19.5 × 5–7.5 μm, mean ± SD = 15.4 ± 1.3 × 6.5 ± 0.5 μm).
Colletotrichum folicola Q. Tan and C.X. Luo, sp. nov. (Figure 14).
MycoBank Number: MB843363.
Etymology: Referring to the host organ from which the fungus was collected.
Figure 13.
Biological characteristics of Colletotrichum karsti. (
a
,
b
) Front and back view of six-day-old
PDA culture; (
c
) conidiomata; (
d
) conidia; (
e
) appressoria; (
f
) conidiophores; (
g
) ascomata; (
h
,
i
) asci;
(j) ascospores ((a–j) isolate YNHH 3-1). Scale bars: (c) = 200 µm; (d–j) = 20 µm.
Colletotrichum folicola Q. Tan and C.X. Luo, sp. nov. (Figure 14).
J. Fungi 2022,8, 313 26 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 26 of 34
Type: China, Yunnan Province, Honghe City, on leaves of Prunus persica cv.
Hongxue, August 2017, Q. Tan. Holotype YNHH 10-1, Ex-type culture CCTCC M
2020345.
Figure 14. Biological characteristics of Colletotrichum folicola. (a,b) Front and back view of
six-day-old PDA culture; (c,d) conidiomata; (e) setae; (f) conidia; (g) appressoria; (h) conidiophores
((a–h) isolate YNHH 10-1). Scale bars: (c,d) = 200 μm; (e–g) = 20 μm; (h,i) = 10 μm.
Sexual morphs were not observed. Asexual morphs developed on PDA. Vegetative
hyphae were hyaline, smooth-walled, septate, and branched. Chlamydospores were not
observed. Conidiomata acervular, conidiophores, and setae formed on hyphae or brown
to black stromata. Conidiomata color ranged from yellow to grayish-yellow to light
brown. Setae were medium brown to dark brown, smooth-walled, 2–6 septa, 50–140 μm
long, base cylindrical, 2.5–4.5 μm in diameter at the widest part, with tip acute. Conidi-
ophores were hyaline to pale brown, smooth-walled, septate, and up to 55 μm long. Co-
nidiogenous cells were hyaline, cylindrical, 12.3−14.5 × 4.4–6.3 μm, with an opening of
1.8–2.5 μm. Conidia were straight, hyaline, aseptate, cylindrical, and had a round end,
12.3−15.4 × 5.6–7.8 μm, mean ± SD = 13.6 ± 0.1 × 6.5 ± 0.3 μm, L/W ratio = 2.1. Appressoria
Figure 14.
Biological characteristics of Colletotrichum folicola. (
a
,
b
) Front and back view of six-day-old
PDA culture; (
c
,
d
) conidiomata; (
e
) setae; (
f
) conidia; (
g
) appressoria; (
h
) conidiophores ((
a
–
h
) isolate
YNHH 10-1). Scale bars: (c,d) = 200 µm; (e–g) = 20 µm; (h,i) = 10 µm.
MycoBank Number: MB843363.
Etymology: Referring to the host organ from which the fungus was collected.
Type: China, Yunnan Province, Honghe City, on leaves of Prunus persica cv. Hongxue,
August 2017, Q. Tan. Holotype YNHH 10-1, Ex-type culture CCTCC M 2020345.
Sexual morphs were not observed. Asexual morphs developed on PDA. Vegetative
hyphae were hyaline, smooth-walled, septate, and branched. Chlamydospores were not
observed. Conidiomata acervular, conidiophores, and setae formed on hyphae or brown to
black stromata. Conidiomata color ranged from yellow to grayish-yellow to light brown.
Setae were medium brown to dark brown, smooth-walled, 2–6 septa, 50–140
µ
m long, base
cylindrical, 2.5–4.5 µm in diameter at the widest part, with tip acute. Conidiophores were
J. Fungi 2022,8, 313 27 of 34
hyaline to pale brown, smooth-walled, septate, and up to 55
µ
m long. Conidiogenous cells
were hyaline, cylindrical, 12.3
−
14.5
×
4.4–6.3
µ
m, with an opening of 1.8–2.5
µ
m. Conidia
were straight, hyaline, aseptate, cylindrical, and had a round end, 12.3
−
15.4
×
5.6–7.8
µ
m,
mean
±
SD = 13.6
±
0.1
×
6.5
±
0.3
µ
m, L/W ratio = 2.1. Appressoria were single, dark
brown, elliptical to clavate, 5.6–13.7
×
4.0
−
8.2
µ
m, mean
±
SD = 8.4
±
0.5
×
5.9
±
0.1
µ
m,
L/W ratio = 1.4.
Culture characteristics: Colonies on PDA attained 16–21 mm diameter in three days at
25
◦
C and 7–10 mm diameter in three days at 30
◦
C; greenish-black, white at the margin,
and aerial mycelium scarce.
Additional specimens examined: China, Yunnan Province, Honghe City, on leaves of
Prunus persica cv. Hongxue, August 2017, Q. Tan, living culture YNHH 2-2.
Notes: Colletotrichum folicola is phylogenetically most closely related to C. citrus-medicae
(Figure 6). The PHI test (
Φ
w = 1) revealed no significant recombination between C. folicola
and C. citrus-medicae (Figure 7), which was described from diseased leaves of Citrus medica
in Kunming, Yunnan Province, China [
54
]. C. folicola is different from C. citrus-medicae
holotype isolate HGUP 1554 in ITS (with 99.04% sequence identity), GAPDH (99.13%),
CHS-1 (98.44%), and HIS3 (99.72%). The sequence data of ACT do not separate the two
species. In terms of morphology, C. folicola differs from C. citrus-medicae by having setae,
smaller conidia (12.3
−
15.4
×
5.6
−
7.8
µ
m vs. 13.5–17
×
5.5–9
µ
m), longer appressoria
(5.6
−
13.7
×
4.0
−
8.2
µ
m vs. 6–9.5
×
5.5
−
8.5
µ
m), and colonies that are greenish-black
rather than white and pale brownish as in C. citrus-medicae.
3.3. Pathogenicity Tests
Pathogenicity tests were conducted to confirm Koch’s postulates on fruit and leaves
for all species identified (Table S4; Figures 15 and 16). Colletotrichum species collected in
this study showed high diversity in virulence. C. nymphaeae,C. fioriniae,C. fructicola, and
C. siamense, which were already reported to be pathogens of peaches, were pathogenic on
both peach leaves and fruit. C. fructicola and C. siamense from the C. gloeosporioides species
complex were more virulent compared to species from the C. acutatum species complex.
Interestingly, C. folicola and C. karsti showed tissue-specific pathogenicity. Isolates of these
two species were all collected from leaves, and mainly infected leaves in the pathogenicity
test. C. folicola did not infect peach fruit at all, and the size of lesions on leaves was
comparably small (0.20
±
0.06 cm). C. karsti did infect peach fruit, but the infection rate was
only around 20% (7/36 isolates) and the size of lesions was
0.06 ±0.01 cm
. In contrast, the
infection rate on leaves was 63.9% (23/36 isolates) and the lesion size was
0.35 ±0.13 cm
.
Isolates of C. godetiae collected from peach leaves in Yunnan province were virulent on
both leaves and fruit, with the leaf and fruit infection rates and lesion diameters being
88.3% (53/60 isolates) and 0.54
±
0.05 cm and 90% (54/60 isolates) and
0.50 ±0.17 cm
,
respectively (Table S4; Figure 16).
J. Fungi 2022,8, 313 28 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 29 of 35
Figure 15. Symptoms of peach fruits and leaves induced by inoculation of spore suspensions of
seven Colletotrichum spp. after six days at 25 °C. (a) Symptoms resulting from H2O, isolates HBYC
Figure 15.
Symptoms of peach fruits and leaves induced by inoculation of spore suspensions of
seven Colletotrichum spp. after six days at 25
◦
C. (
a
) Symptoms resulting from H
2
O, isolates HBYC 1,
JXJA 6, and YNHH 1-1 (
left
to
right
). (
b
) Symptoms resulting from isolates GDHY 10-1, SDQD 10-1,
YNHH3-1, and YNHH10-1 (left to right).
J. Fungi 2022,8, 313 29 of 34
J. Fungi 2022, 8, x FOR PEER REVIEW 30 of 35
1, JXJA 6, and YNHH 1-1 (left to right). (b) Symptoms resulting from isolates GDHY 10-1, SDQD
10-1, YNHH3-1, and YNHH10-1 (left to right).
Figure 16. Lesion size on peach fruit and leaves of seven Colletotrichum spp. in the six days after
inoculation. C. nymphaeae isolates FJFZ 1, HBJM 1-1, HBWH 3-2, HBYC 1, SCCD 1; C. fioriniae
isolates GZTR 7-1, JXJA 1, JXJA 6, ZJLS 1, ZJLS 11-1; C. godetiae isolates YNHH 1-1, YNHH 2-1,
YNHH 4-1, YNHH 7-2, YNHH 9-1; C. fructicola isolates GDHY 10-1, GDSG 1-1, GDSG 5-1, GZTR
10-1, GZTR 13-1; C. siamense isolates HBSJZ 1-1, HBSJZ 3-1, HBSJZ 5-1, HBSJZ 7-1, SDQD 10-1; C.
karsti isolates YNHH 3-1, YNHH 3-2, YNHH 5-2; C. folicola isolates YNHH 2-2, YNHH 10-1.
Letters over the error bars indicate a significant difference at the p = 0.05 level. Capital letters refer
to fruit and lowercase letters to leaves.
4. Discussion
This study is the first large-scale investigation of Colletotrichum species causing
anthracnose fruit and leaf diseases in peaches in China. The most common
Colletotrichum species were C. nymphaeae and C. fioriniae of the C. acutatum species
complex and C. fructicola and C. siamense of the C. gloeosporioides species complex.
The same species were also identified in the southeastern USA [17,21,22], where a shift
over time appeared to favor C. gloeosporioides species complex in South Carolina. The
authors speculated that inherent resistance of C. acutatum to benzimidazole fungicides
(MBCs) may have given this species complex a competitive advantage when MBCs were
frequently used [22]. As MBCs were replaced by other fungicides (including quinone
outside inhibitors and demethylation inhibitors), that competitive advantage may have
disappeared and C. gloeosporioides species may have increased in prevalence [22,55]. In
support of this hypothesis is previous research showing a higher virulence of C.
gloeosporioides on peaches, pears, and apples compared to C. acutatum [8,56,57]. Also,
this study and others show that the C. gloeosporioides species complex may be better
adapted to the hot South Carolina climate compared to the C. acutatum species complex
[3]. MBCs are still popular fungicides in Chinese peach production regions. Therefore, it
is possible that the dominance of C. acutatum species complex, specifically C. nymphaeae
is, at least in part, a result of fungicide selection.
The high prevalence of C. nymphaeae in Chinese peach orchards is consistent with
other local studies reporting the same species affecting a wide variety of other fruit crops
in China. For example, C. nymphaeae was reported in Sichuan province on blueberries
Figure 16.
Lesion size on peach fruit and leaves of seven Colletotrichum spp. in the six days after
inoculation. C. nymphaeae isolates FJFZ 1, HBJM 1-1, HBWH 3-2, HBYC 1, SCCD 1; C. fioriniae isolates
GZTR 7-1, JXJA 1, JXJA 6, ZJLS 1, ZJLS 11-1; C. godetiae isolates YNHH 1-1, YNHH 2-1, YNHH 4-1,
YNHH 7-2, YNHH 9-1; C. fructicola isolates GDHY 10-1, GDSG 1-1, GDSG 5-1, GZTR 10-1, GZTR
13-1; C. siamense isolates HBSJZ 1-1, HBSJZ 3-1, HBSJZ 5-1, HBSJZ 7-1, SDQD 10-1; C. karsti isolates
YNHH 3-1, YNHH 3-2, YNHH 5-2; C. folicola isolates YNHH 2-2, YNHH 10-1. Letters over the error
bars indicate a significant difference at the p= 0.05 level. Capital letters refer to fruit and lowercase
letters to leaves.
4. Discussion
This study is the first large-scale investigation of Colletotrichum species causing anthrac-
nose fruit and leaf diseases in peaches in China. The most common Colletotrichum species
were C. nymphaeae and C. fioriniae of the C. acutatum species complex and C. fructicola and
C. siamense of the C. gloeosporioides species complex. The same species were also identified in
the southeastern USA [
17
,
21
,
22
], where a shift over time appeared to favor C. gloeosporioides
species complex in South Carolina. The authors speculated that inherent resistance of
C. acutatum to benzimidazole fungicides (MBCs) may have given this species complex a
competitive advantage when MBCs were frequently used [
22
]. As MBCs were replaced
by other fungicides (including quinone outside inhibitors and demethylation inhibitors),
that competitive advantage may have disappeared and C. gloeosporioides species may have
increased in prevalence [
22
,
55
]. In support of this hypothesis is previous research showing
a higher virulence of C. gloeosporioides on peaches, pears, and apples compared to C. acuta-
tum [
8
,
56
,
57
]. Also, this study and others show that the C. gloeosporioides species complex
may be better adapted to the hot South Carolina climate compared to the C. acutatum
species complex [
3
]. MBCs are still popular fungicides in Chinese peach production regions.
Therefore, it is possible that the dominance of C. acutatum species complex, specifically
C. nymphaeae is, at least in part, a result of fungicide selection.
The high prevalence of C. nymphaeae in Chinese peach orchards is consistent with
other local studies reporting the same species affecting a wide variety of other fruit crops
in China. For example, C. nymphaeae was reported in Sichuan province on blueberries and
loquats [
58
,
59
], in Hubei province on strawberries and grapevines [
60
,
61
], and in Zhejiang
J. Fungi 2022,8, 313 30 of 34
province on pecans [
62
]. Internationally, it is one of the most common species affecting
pome fruits, stone fruits, and small fruits [23,63,64].
C. godetiae,C. karsti, and C. folicola were reported on peaches for the first time. The
three species were geographically isolated and only present in Yunnan province. Rare
occurrences of Colletotrichum species have also been formerly observed on peaches, i.e.,
C. truncatum was only found in one of many orchards examined in South Carolina, USA [
25
].
C. godetiae and C. karsti are well-known pathogens of fruit crops. C. godetiae was reported
to cause disease on apples, strawberries, and grapes [
65
–
68
], while C. karsti was reported
to affect apples and blueberries [
69
,
70
]. It is, therefore, possible that these pathogens
migrated from other hosts into Yunnan province peach orchards. The observed occurrence,
however, does point to either a rather rare host transfer event or to environmental conditions
that favor these species. Yunnan province is located in southwestern China and peach
production is popular in the Yunnan–Guizhou high plateau, a region with low latitude and
high altitude [
71
]. The complicated local topography and diverse climate lead to highly
abundant biodiversity [
72
], which may explain the emergence of the new species C. folicola.
As mentioned above, regional differences in Colletotrichum species composition in
commercial orchards may be influenced by fungicide selection pressure. For example,
C. acutatum is less sensitive to benomyl, thiophanate-methyl, and other MBC fungicides
compared with C. gloeosporioides [
56
,
73
,
74
]. Meanwhile, all C. nymphaeae strains in this
study have been confirmed to be resistant to carbendazim (MBC) [
75
]. C. nymphaeae
was reported to be less sensitive to demethylation inhibitor (DMIs) fungicides (flutri-
afol and fenbuconazole) compared with C. fioriniae,C. fructicola, and C. siamense [
21
] and
C. gloeosporioides was reported to be inherently tolerant to fludioxonil [
76
,
77
]. Most of the
peach farms in China are small and there is vast diversity in the approaches to managing
diseases. However, MBC (i.e., carbendazim and thiophanate-methyl) fungicides are com-
monly used to control peach diseases, followed by DMIs (i.e., difenoconazole). Whether
fungicide selection had an impact on the Colletotrichum species distribution is unknown,
but the high prevalence of C. acutatum species complex and their resilience to MBCs (and,
in the case of C. nymphaeae, to DMIs) would allow for such a hypothesis.
In conclusion, this study provides the morphological, molecular, and pathological
characterization of seven Colletotrichum spp. occurring on peaches in China. This is of
great significance for the prevention and control of anthracnose disease in different areas
in China.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/jof8030313/s1, Table S1: Primers used in this study, with sequences,
annealing temperature, and sources [
51
,
78
–
85
]; Table S2: Isolates of seven Colletotrichum species
collected from peaches in China, with details about host tissue, location, and GenBank accession
number; Table S3: The sizes of conidia, appresoria, ascospores, and mycelial growth rate of the
representative isolates of Colletotrichum spp. obtained in this study; Table S4: Infection rates of seven
Colletotrichum spp. inoculated on peach fruit and leaves.
Author Contributions:
Conceptualization, Q.T., G.S. and C.-X.L.; methodology, Q.T., C.C., W.-X.Y.,
L.-F.Y. and C.-X.L.; software, Q.T. and C.C.; validation, Q.T. and C.-X.L.; formal analysis, Q.T. and
G.S.; investigation, Q.T., L.-F.Y. and C.-X.L.; data curation, Q.T. and C.-X.L.; writing—original draft
preparation, Q.T., G.S. and C.-X.L.; writing—review and editing, G.S., C.C. and C.-X.L.; visualization,
Q.T. and C.C.; supervision, W.-X.Y. and C.-X.L.; project administration and funding acquisition,
C.-X.L. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the China Agriculture Research System of the Ministry of
Finance and the Ministry of Agriculture and Rural Affairs (CARS-30-3-03), and the Fundamental
Research Funds for the Central Universities (No. 2662020ZKPY018).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
J. Fungi 2022,8, 313 31 of 34
Data Availability Statement:
Alignments generated during the current study are available from
TreeBASE (http://treebase.org/treebase-web/home.html; study 29227). All sequence data are
available in the NCBI GenBank, following the accession numbers in the manuscript.
Acknowledgments:
We sincerely thank the reviewers for their contributions during the
revision process.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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