plants
Article
Two Newly Identified Colletotrichum Species Associated with
Mango Anthracnose in Central Thailand
Chainarong Rattanakreetakul 1 , Pisut Keawmanee 1 , Santiti Bincader 2 , Orarat Mongkolporn 3 ,
Vipaporn Phuntumart 4 , Sotaro Chiba 5 and Ratiya Pongpisutta 1, *
1
2
3
4
5
*
Citation: Rattanakreetakul, C.;
Keawmanee, P.; Bincader, S.;
Mongkolporn, O.; Phuntumart, V.;
Chiba, S.; Pongpisutta, R. Two Newly
Identified Colletotrichum Species
Associated with Mango Anthracnose
in Central Thailand. Plants 2023, 12,
1130. https://doi.org/10.3390/
plants12051130
Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University,
Nakhon Pathom 73140, Thailand
Program Plant Science, Agricultural Technology and Agro-Industry Faculty, Rajamangala University of
Technology Suvarnabhumi, Phra Nakhon Si Ayutthaya 13000, Thailand
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University,
Nakhon Pathom 73140, Thailand
Department of Biological Sciences, 129 Life Sciences Building, Bowling Green State University,
Bowling Green, OH 43403, USA
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Correspondence: ratiya.p@ku.th; Tel.: +66-34-351-890
Abstract: Anthracnose caused by Colletotrichum spp. is one of the major problems in mango production worldwide, including Thailand. All mango cultivars are susceptible, but Nam Dok Mai
See Thong (NDMST) is the most vulnerable. Through a single spore isolation method, a total of
37 isolates of Colletotrichum spp. were obtained from NDMST showing anthracnose symptoms. Identification was performed using a combination of morphology characteristics, Koch’s postulates, and
phylogenetic analysis. The pathogenicity assay and Koch’s postulates on leaves and fruit confirmed
that all Colletotrichum spp. tested were causal agents of mango anthracnose. Multilocus analysis
using DNA sequences of internal transcribed spacer (ITS) regions, β-tubulin (TUB2), actin (ACT), and
chitin synthase (CHS-1) was performed for molecular identification. Two concatenated phylogenetic
trees were constructed using either two-loci of ITS and TUB2, or four-loci of ITS, TUB2, ACT, and
CHS-1. Both phylogenetic trees were indistinguishable and showed that these 37 isolates belong to
C. acutatum, C. asianum, C. gloeosporioides, and C. siamense. Our results indicated that using at least
two loci of ITS and TUB2, were sufficient to infer Colletotrichum species complexes. Of 37 isolates,
C. gloeosporioides was the most dominant species (19 isolates), followed by C. asianum (10 isolates),
C. acutatum (5 isolates), and C. siamense (3 isolates). In Thailand, C. gloeosporioides and C. acutatum
have been reported to cause anthracnose in mango, however, this is the first report of C. asianum and
C. siamense associated with mango anthracnose in central Thailand.
Keywords: Colletotrichum; Mangifera indica L.; species identification; multilocus phylogeny
Academic Editor: Rishi R. Burlakoti
Received: 6 December 2022
Revised: 24 February 2023
Accepted: 26 February 2023
Published: 2 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Mango production has expanded to more than 100 countries, with around 44.6 M tons
annually since 2018 [1]. Thailand is a major producer and contributes to almost 8% of
the global mango production [1]. Because of its flavor and texture, Nam Dok Mai See
Thong (NDMST) has become the most popular mango cultivar in Thailand. It is also an
early-midseason cultivar, which means that it has the potential to produce fruit all year
round [2,3]. Anthracnose caused by Colletotrichum spp. is a significant economic problem
in both mango orchards and postharvest storage [3–8]. The pathogen infects not only
fruit but also inflorescences, flowers, and leaves. During the flowering stage, especially
under high humidity, a disease incidence of 100% has been observed [4,9–12]. Young leaves
that emerge during rainy periods are also prone to anthracnose infection. Anthracnose
symptoms on leaves appear as small and dark brown spots often surrounded with chlorotic
Plants 2023, 12, 1130. https://doi.org/10.3390/plants12051130
https://www.mdpi.com/journal/plants
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haloes and irregular margins [2,6,9,12,13]. Leaf lesions usually remain small, however, under disease-favorable conditions, the lesions can enlarge and fuse together to form irregular
patches [2,4,5,14]. Other symptoms include premature leaf drop and twig dieback [4,15,16].
Mango fruit at any stage can be infected [5,17–20]. Mummification was usually observed
in young fruit, while no symptom was observed in mature unripe fruit. On ripe fruit,
dark brown irregular lesions appear, which gradually increase in irregular size and under
favorable conditions, salmon to orange fungal conidial masses can be observed on the lesions [4,5,14,21]. The application of fungicides is a common practice to control anthracnose
in mango orchards in Thailand, although it is often unsuccessful. The inefficient fungicide application is probably due to the emergence of new Colletotrichum species and/or
fungicide resistance of the pathogens.
Accurate identification of species is a necessary starting point for the effective management of anthracnose disease. Currently, three Colletotrichum species; C. acutatum, C. boninense, and C. gloeosporioides have been reported to cause anthracnose in mango [22–24].
However, C. gloeosporioides species complex, which are ubiquitous fungal pathogens in
tropical and sub-tropical areas, has not been reported in Thailand. The host-association and
morphological characteristics that have been used to identify Colletotrichum species [25,26]
are insufficient to distinguish these pathogens at the species level due to the limited number of morphological characters and the pathogens are not host specific. A polyphasic
approach, multilocus phylogenetic analysis in conjunction with recognizable phenotypic
characters, has been recommended to accurately identify species within the Colletotrichum
genus [27,28]. According to Weir et al. [27], internal transcribed spacer (ITS) sequences alone
are not reliable to distinguish different species within the C. gloeosporioides species complex.
Additional recommended loci for the identification of the Colletotrichum species included
actin (ACT), calmodulin (CAL), chitin synthase-1 (CHS-1), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and β-tubulin (TUB2) [7,9,23,26,28–30]. The identification of
C. gloeosporioides species complex has not been fully investigated in mango anthracnose
in Thailand. The identification of C. gloeosporioides species complex has not been fully
investigated in mango anthracnose in Thailand. We hypothesize that the C. gloeosporioides
species complex could be a potential source of new Colletotrichum species. This study
used an integrative approach of a morphological assay, multilocus phylogenetic analysis,
and pathogenicity test to identify species of the Colletotrichum infecting mango NDMST in
Thailand. Here, we report for the first time that C. asianum and C. siamense are casual fungi
causing anthracnose in mango grown in central Thailand.
2. Results
2.1. Fungal Isolates
Inflorescence, leaves, and fruit showing typical anthracnose symptoms were collected
from orchards located in Chachoengsao, Phichit and Ratchaburi. Visual anthracnose symptoms were inflorescence blight (Figure 1a,b), small brown to black spots and irregularly
shaped lesions with brown to black necrotic lesions on leaves (Figure 1c,d). Symptoms
on ripe fruit were small black circular spots, irregular and necrotic sunken brown to black
lesions. On severely infected fruit, extensive fruit rot was observed (Figure 1e), under high
humidity, bright orange to pale colored spore masses can be seen (Figure 1f). Infected premature fruit were usually dropped from the trees. A total of 37 fungal isolates resembling
Colletotrichum spp. were obtained (Table 1).
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Figure 1. Visual symptoms of anthracnose caused by Colletotrichum spp. on mango tissues of NDMST
cultivar; blight on inflorescence (a,b), irregular necrotic lesions on leaves (c,d) and irregular and
sunken necrotic lesion on fruit (e) with abundant spore masses (f).
Table 1. Collection of the Colletotrichum isolates with their geographical information. All isolates
were from the NDMST cultivar.
Geographic Coordinates 1
Location of Origin
Plaeng Yao 1, Chachoengsao −
13◦ 36′ 28.6′′
Plaeng Yao 2, Chachoengsao
13◦ 36′ 42.2′′ N 101◦ 17′ 41.6′′ E
N
101◦ 17′ 53.5′′
−1
Isolate Code
E
–
–
–
Source
–
CS001
CS002
CS003
Fruit
Fruit
–
Fruit
CS004
CS005
CS006
CS007
Fruit
Leaf
Leaf
Inflorescence
Plaeng Yao 2, Chachoengsao
13◦ 36′ 50.2′′ N 101◦ 17′ 24.8′′ E
CS008
CS009
CS010
Fruit
Fruit
Leaf
Sak Lek 1, Phichit
16◦ 28′ 20.9′′ N 100◦ 33′ 42.4′′ E
PC001
PC002
PC003
PC004
PC005
PC006
PC007
Fruit
Fruit
Fruit
Leaf
Fruit
Leaf
Fruit
−1
–
Sak Lek 2, Phichit
16◦ 28′ 41.7′′ N 100◦ 33′ 52.4′′ E
–
Mueang Ratchaburi, Ratchaburi
13◦ 35′ 50.0′′ N 99◦ 49′ 57.1′′ E
–
PC008
PC009
–
PC010
PC011
PC012
Leaf
Fruit
Leaf
Leaf
Leaf
RB001
RB002
Fruit
Leaf
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Table 1. Cont.
Location of Origin
Geographic Coordinates 1
Paktho, Ratchaburi
13◦ 24′ 47.5′′
Bang Phae, Ratchaburi
1
Isolate Code
Source
E
RB003
RB004
RB005
RB006
RB007
RB008
RB009
RB010
RB011
RB012
Fruit
Inflorescence
Fruit
Fruit
Fruit
Fruit
Fruit
Fruit
Fruit
Fruit
13◦ 39′ 50.5′′ N 99◦ 57′ 54.4′′ E
RB013
RB014
RB015
Fruit
Fruit
Fruit
N
99◦ 45′ 32.9′′
Geographic location was detected by GPS status version 8.0.170.
2.2. Morphology-Based Identification
Based on colony morphology and conidia characteristics, the Colletotrichum isolates
were classified into two groups. The first group contained five isolates that were similar to C. acutatum. The colony grown on PDA appeared white to grey and the reverse side of the colony was pale ochreous. The mycelial growth rates at day 3 ranged
from 3.33–3.77 mm day−1 (average = 3.56 mm day−1 ). Spore masses were bright orange,
conidia were hyaline, aseptate, straight, apex obtuse, and no setae. The sizes of conidia ranged from 3.38–6.17 µm (average = 4.43 µm) in width and from 10.96–19.88 µm
(average = 14.8 µm) in length. Appressoria were clavate, long and irregular shapes, pale to
dark brown in color. The diameter of appressoria ranged from 3.86–6.95 (average = 5.45 µm)
in width and ranged from 5.97–11.20(8.36) µm (Table S1, Figure 2).
The second group contained 32 isolates with similarity to C. gloeosporioides (some of
these isolates were later classified as C. asianum or C. siamense when DNA markers were
integrated, described below). The colony grown on PDA showed a great variation in
color from white, greenish to grayish, pale yellowish to dark grey, while the reverse sides
were dark green (Figure 2). The mycelial growth rates also varied from 2.13 to 5.43 mm
(average = 4.01 mm day−1 ). Conidia were hyaline, aseptate, straight cylindrical, rounded
at the apex end and conspicuous hilum at basal end, and no setae. The sizes of conidia
ranged from 3.60–7.07 µm (average = 5.03 µm) in width and ranged from 10.33–19.95 µm
(average = 14.24 µm) in length (Table S1, Figure 2). Spore masses were pale, salmonorange to bright orange colors. Various shapes of appressoria of clavate, long clavate,
occasionally irregular, pale to dark brown in color were observed (Table S1, Figure 2). The
size of the appressoria ranged from 4.42–8.09 µm (average = 5.82 µm) × 5.53–11.59 µm
(average = 8.72 µm) (Table S1, Figure 2).
2.3. DNA Marker-Based Identification
All DNA sequences of ACT, CHS-1, ITS, and TUB2 were subjected to BLASTn (Table S2).
Sequences of ex-type or epitype strains of Colletotrichum species (Table S2) were selected for
phylogenetic analysis. We firstly constructed a phylogenetic tree using two DNA markers:
ITS and TUB2. A total of 47 isolates, including ten isolates of ex-type and ex-epitype or
epitype strains, and Leptosphaeria veronicae CBS145.84 was used as an outgroup (Figure 3).
The topology of the ML tree and Bayesian tree were identical, therefore, only the ML
tree is shown. A discrete Gamma distribution was used to estimate the divergence and
evolutionary rate (+G, parameter = 7.1107) (Figure 3). The 37 isolates were assigned to four
species clades on the maximum likelihood (ML) tree. Of these, 19 isolates formed a clade
that was closely related to C. gloeosporioides strain (ICMP17821), showing 0.13 posterior
probability with a bootstrap value of 100%, three isolates were clustered with C. siamense
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(ICMP12567) with 0.01 posterior probability and a bootstrap value of 100%; ten isolates
clustered with C. asianum (ICMP18696) with 0.00 posterior probability and a bootstrap value
of 97%, and five isolates were grouped with C. acutatum (CBS144.29) with 0.02 posterior
probability and a bootstrap value of 100%.
Figure 2. Morphological characteristics of colonies grown on PDA at day 3, pictures were taken from
the front and the back of the plates. Appressoria were measured at day 4 and conidia were measured
at day 5 after incubation. Shown here are the representatives of each species. C. acutatum PC011 (a),
C. asianum RB001 (b), C. gloeosporioides CS005 (c), and C. siamense RB003 (d).
The incongruence length difference (ILD) test showed that the ACT, CHS-1, ITS, and
TUB2 were homogeneous. Therefore, the concatenated sequences of four markers; ACT,
CHS-1, ITS, and TUB2 were used to generate a phylogenetic tree with a total of 81 isolates,
including 44 isolates of ex-type and ex-epitype or epitype strains. The topology of the
ML tree was consistent with that of the Bayesian tree, and therefore only the ML tree is
shown (Figure 4). Among the 37 isolates, ten isolates were clustered with C. asianum strains
(ICMP 18580, ICMP 18696, NN8, WM52, and NN19) showing 0.97 posterior probability
and with bootstrap values of 99%; three isolates formed a clade with C. siamense strains
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(ICMP18578, ICMP17795, ICMP18121, ICMP12567, and ICMP18574) with 0.97 posterior
probability with bootstrap values of 77%; and 19 isolates clustered with the C. gloeosporioides
strain (ICMP17821) with 1.00 posterior probability with bootstrap values of 99%. Five isolates were clustered with C. acutatum (CBS112996, IMI223120, IMI216370, CBS144.29, and
CBS979.69) with 0.85 posterior probability with bootstrap values of 99%.
Figure 3. The maximum likelihood tree based on a concatenated data set of ITS and TUB2 sequences
of 47 Colletotrichum isolates, ex-type/epitype, were retrieved from GeneBank. Numbers on the node
are bootstrap values (left) and posterior probability (right). Colletotrichum isolates from this study are
in grey boxes. The scale bar shows the number of substitutions per site.
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Figure 4. The maximum likelihood tree based on the concatenated data set of ACT, CHS-1, ITS, and
TUB2 sequences from a total of 81 Colletotrichum species with the ex-type/epitype were retrieved
from GeneBank. Numbers on the node are bootstrap values (left) and posterior probability (right).
Colletotrichum isolates from this study are in red. The scale bar shows the number of substitutions
per site.
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2.4. Pathogenicity Test
A mycelial plug of a 5-day-old culture was inoculated on unwounded fruit and
leaves. Typical anthracnose lesions were observed around the inoculation sites. The
anthracnose lesions on fruit enlarged faster than those on leaves on day 5 after inoculation
(Table 2 and Figure 5). No lesions were observed on the control fruit or leaves (Figure 5).
On inoculated fruit, C. asianum produced the largest lesions that differed significantly from
other species with the average LD of 8.16 cm, followed by the LD means of C. gloeosporioides,
C. siamense, and C. acutatum at 8.07, 7.81, and 7.61 cm, respectively (Table 2 and Figure 6).
Table 2. Lesion sizes on NDMST mango fruit and leaves after inoculation by the 37 isolates of
Colletotrichum spp.
Isolate Code
Taxon
CS001
CS002
CS003
CS004
CS005
CS006
CS007
CS008
CS009
CS010
PC001
PC002
PC003
PC004
PC005
PC006
PC007
PC008
PC009
PC010
PC011
PC012
RB001
RB002
RB003
RB004
RB005
RB006
RB007
RB008
RB009
RB010
RB011
RB012
RB013
RB014
RB015
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. asianum
C. siamense
C. asianum
C. asianum
C. asianum
C. acutatum
C. acutatum
C. gloeosporioides
C. gloeosporioides
C. gloeosporioides
C. acutatum
C. acutatum
C. asianum
C. gloeosporioides
C. siamense
C. gloeosporioides
C. asianum
C. siamense
C. asianum
C. gloeosporioides
C. gloeosporioides
C. asianum
C. asianum
C. asianum
C. gloeosporioides
C. acutatum
C. gloeosporioides
Lesion Diameter (cm) *
Fruit
Leaves
9.30a
9.07ab
7.97e–j
9.00abc
8.62b–e
7.85f–k
8.53b–f
8.32c–h
8.50b–f
8.38b–g
8.52b–f
8.72a–d
8.67a–e
8.48b–f
8.73a–d
6.88m
8.38b–g
7.47i–m
7.53i–m
7.18klm
8.03d–i
7.48i–m
7.67h–l
7.48i–m
7.48i–m
7.63h–l
7.65h–l
7.23klm
7.50i–m
7.73g–l
8.40b–g
8.13d–i
7.88f–k
8.32c–h
7.28j–m
7.27j–m
7.08lm
0.33gh
0.58c–h
0.45d–h
0.75b–h
1.12bc
1.03b–e
0.65b–h
0.80b–h
0.43e–h
0.48c–h
0.60c–h
0.58c–h
0.82b–h
1.10bcd
0.93b–g
0.32gh
0.25h
0.82b–h
0.93b–g
0.35fgh
0.45d–h
0.40e–h
0.37fgh
0.72b–h
0.68b–h
0.67b–h
0.82b–h
2.48a
0.93b–g
0.73b–h
0.88b–h
1.28b
1.28b
1.03b–e
0.80b–h
0.50c–h
1.00b–f
* Values with the same column followed by different common letters mean that they are significantly different
based on variance with the least significant difference test at p = 0.05.
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Figure 5. Pathogenicity assay of four different Colletotrichum species on NDMST mango; visual
anthracnose symptoms developed on fruit (a), leaves (b) at 5 days after inoculation, and appressoria
and spores developed on leaves at 24 h after inoculation (c). The test was performed on all isolates;
only a representative of each species is shown here.
isolated from the infected tissues and confirmed by Koch’s postulates to have
Figure 6. Box plots showing the variation of disease lesion diameter amongst four Colletotrichum
species associated with mango anthracnose at 5 days after inoculation with a mycelial plug on
unwounded NDMST mango fruit and leaves. Vertical lines are median. Means followed by the
different common letter are significantly different according to the Tukey-test.
Similar results were observed on inoculated leaves; C. siamense showed the largest
lesions that differed significantly from other species with the average LD of 1.25 cm, whereas
the LD means of C. asianum, C. gloeosporioides and C. acutatum were 0.92, 0.71, and 0.38 cm,
–
respectively (Table 2 and Figure 6). After the pathogenicity test, all the Colletotrichum spp.
were re-isolated from the infected tissues and confirmed by Koch’s postulates to have
–
identical morphological characteristics as the original isolates.
and Koch’s pos-
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3. Discussion
It has been widely accepted that high genetic variation within Colletotrichum species
complexes exists due to their wide host range and diverse environments [23–27]. Mango anthracnose has been reported to associate with different species of Colletotrichum. This study
aimed to accurately identify the species of the Colletotrichum–mango system in Thailand,
using a combination of morphology, multilocus sequence analyses, and Koch’s postulates.
A total of 37 Colletotrichum species were isolated from mango anthracnose disease from
orchards located in central Thailand. When morphological characteristics such as colony
color, growth rate, size, and shape of conidia and appressoria were used, 32 isolates were
identified as C. acutatum, and five isolates were identified as C. gloeosporioides. It is generally
accepted that morphological characteristics alone are not sufficient for species identification since variation in traits among species can be similar under different environments,
therefore the multilocus phylogenetic approach was integrated to aid in taxonomy.
Several DNA markers have been developed to identify Colletotrichum isolates. A multilocus sequence analysis using ITS, CAL, or TUB2 identified C. alienum, C. fructicola, or
C. tropicale from other Colletotrichum species [27]. Similarly, a study of lupin anthracnose
by Alkemade et al. [31] showed that 39 out of 50 isolates belonged to Colletotrichum lupini.
The authors also used the combination of multilocus analysis (ITS, TUB2, GAPDH, and
APN/MAT1) and morphological characteristics to support their taxonomic classification
of Colletotrichum species complex. This study used phenotypic characters, DNA markers
of ITS, ACT, CHS-1, and TUB2, and Koch’s postulates to confirm that all 37 Colletotrichum
isolates were mango anthracnose pathogens. In addition to the four DNA markers used
in this study, GAPDH has also been used to identify C. acutatum and C. gloeosporioides
complexes by Damm et al. [23] and Weir et al. [27]. In this study, two phylogenetic trees
were constructed using two loci (ITS and TUB2) and four loci (ITS, TUB2, ACT and CHS-1).
The topology of both trees was similar and further identified the Colletotrichum species
into C. acutatum, C. gloeosporioides, C. asianum and C. siamense. These results indicated that
using two loci is sufficient to distinguish these four species of Colletotrichum. Colletotrichum
gloeosporioides and C. acutatum were previously reported in 1979 and 2019 [32]; however,
we have uncovered for the first time that C. asianum and C. siamense caused anthracnose in
mango in Thailand.
Colletotrichum asianum has been reported to be a major species causing mango anthracnose in China [33]. It has been the most common endophytic species of mango in
northeastern Brazil [34] and in many countries around the world, such as Australia, China,
Colombia, Japan, Malaysia, Philippines, Sri Lanka, and Taiwan [11–13,18,19,27,35,36]. Colletotrichum asianum is also capable of causing disease in avocado (Persea americana Mill.) in
Australia [19]. In Thailand, C. asianum has been reported to cause disease in coffee (Coffea
arabica L.) [37] but not in mango until this study.
Colletotrichum siamense has been reported to be a major species causing mango anthracnose in China [33] and in eastern Australia [38]. It has a wide host range in tropical and
subtropical regions and can infect banana (Musa spp.), papaya (Carica papaya L.), dragon
fruit (Hylocereus spp.), guava (Psidium guajava) and avocado [12,18,19,27,33,39–46]. It was
suggested by James et al. [38] that C. siamense is likely to be a common and widespread
saprophyte or endophyte because the authors were able to isolate it from asymptomatic fruit
of other plants, except mango and avocado. In Thailand, similar to C. asianum, C. siamense
has been reported to cause disease in coffee (Coffea arabica L.) [37] but not in mango until
this study.
Among the four species identified, C. gloeosporioides was a major species causing anthracnose in this study. It was found in every orchard that we isolated. The pathogenicity
assay showed that all isolates appeared to be more aggressive on fruit than leaves, probably because fruit contains more sugar that serves as a carbon source for fungal growth.
C. asianum and C. gloeosporioides showed a similar degree of aggressiveness on mango
fruit and produced larger lesions than C. siamense and C. acutatum. The color of lesions
were also slightly different on inoculated fruit, C. asianum produced dark brown spots,
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while the other three species produced lighter brown spots. The pathogenicity on the leaf
showed that C. siamense was the most aggressive compared to other species, possibly due
to species-specific host responses. Further testing using different cultivars of mango should
be employed to verify this hypothesis.
The impact of this study was twofold: firstly, it has proven that a combination of
morphology, DNA multilocus sequence analysis, and Koch’s postulates is a robust identification approach to identify species of Colletotrichum, and secondly, our approach enables
the discovery of previously unreported anthracnose caused by C. asianum and C. siamense,
in Thailand. This discovery raises a concern regarding the cross-infection potential where
the two species can infect different hosts. Therefore, accurate diagnosis is a crucial first step
for disease control and prevention. It supports the current quarantine regulations as well as
establishes strategies for integrated management of anthracnose disease between orchards.
It should be noted that fungal pathogens, although in the same genus, will respond to
fungicides differently. Further study is essential to determine the fungicide sensitivity of
these four Colletotrichum species to help implement the fungicide management strategy.
4. Materials and Methods
4.1. Fungal Isolation
The infected samples of 25 fruit, 10 leaves, and two inflorescences with typical anthracnose symptoms were collected from eight orchards located in central Thailand: Chachoengsao, Phichit, and Ratchaburi, in 2016–2017. The locations of orchards, geographic
coordinates, codes of the isolates, and types of infectious tissues collected are provided
in Table 1. Thirty-seven isolates of Colletotrichum spp. were recovered. The pathogens
were isolated and cultured on potato dextrose agar (PDA) using a tissue transplantation
technique. To obtain pure isolates, mycelial plugs were sub-cultured on fresh PDA, and
incubated at 25 ◦ C under a photoperiod of 12 h light/12 h dark for 5 days [8], followed by
single spore isolation on water agar (WA). The single spore from each isolate was transferred to a new PDA plate and incubated under the same condition. Each colony served as
a single genetic source for further analysis.
4.2. Morphological Characteristics
Each isolate was inoculated with a 6-mm-diameter plug taken from an actively growing edge of a 5-day-old culture on a PDA plate. The culture was incubated under the
same condition mentioned above. Fungal growth and colony diameter were recorded daily
until there were no changes in diameter (day 3). Fifty conidia were randomly selected for
measurement of their length and width at day 5 (conidia were fully matured) under an
Olympus CX31 binocular compound microscope at 400× magnification with the Olympus
CellSens standard software version 1.16 (Olympus Co., Ltd., Tokyo, Japan). Appressoria
were induced using a slide culture technique [47]. Briefly, the isolates were transferred onto
25.4 × 76.2 mm sterile microscope slides (7101 microscope slides, Shandong Harmowell
Trade Co., Ltd., Shanghai, China) and covered with 22 × 22 mm coverslips (Menzel Gläser,
Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA) incubated in a petri dish at 25 ◦ C
until maturation, usually for 4 days. Lengths and widths of 30 appressoria per isolate were
measured under a microscope. This experiment was performed in five replicates.
4.3. DNA Extraction and Molecular Identification
Genomic DNA was extracted from a 5-day-old fungal colony, according to Pongpisutta
et al. [48]. A PCR mixture contained genomic DNA (20 ng), primers (0.48 µM each, Table 3),
Taq polymerase buffer (1×, Thermo Fisher Scientific, Waltham, MA, USA), MgCl2 (2.4 mM),
dNTPs (10 µM each), Taq polymerase (1 U, Thermo Fisher Scientific, Waltham, MA, USA).
PCR was performed in a thermal cycler (Sensoquest GmbH, Göttingen, Germany). The PCR
was programmed as follows: 94 ◦ C for 2 min, 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55–58 ◦ C (Table 3) for 30 s, and extension at 72 ◦ C for 1 min, with a final extension
step of 72 ◦ C for 10 min. The PCR products were visualized on 1.2% agarose gels stained
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with GelStar® and GeneRuler, and 100 bp Plus DNA Ladder (Thermo Fisher Scientific,
Waltham, MA, USA) was used to determine the size of DNA fragments. The PCR products
were sequenced by the 1st Base Laboratory Co., Ltd., Seri Kembangan, Malaysia. A basic
local alignment search, BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on
22 April 2021), was performed to analyze nucleotide sequences in comparison to reference
sequences available in the National Center for Biotechnology Information (NCBI) database.
Table 3. Primers used in this study with sequences and sources.
Annealing
Temperature (◦ C)
References
ATGTGCAAGGCCGGTTTCGC
TACGAGTCCTTCTGGCCCAT
58
[49]
T1
T2
AACATGCGTGAGATTGTAAGT
TAGTGACCCTTGGCCCAGTTG
55
[50]
Chitin synthase 1
CHS-79F
CHS-345R
TGGGGCAAGGATGCTTGGAAGAAG
TGGAAGAACCATCTGTGAGAGTTG
58
[49]
ITS region
ITS5
ITS4
GGAAGTAAAAGTCGTAACAAGG
TCCTCCGCTTATTGATATGC
56
[51]
Gene
Primer
Sequence (5′ -3′ )
Actin
ACT-512F
ACT-783R
β-tubulin
4.4. Phylogenetic Analyses
Multiple sequence alignments were performed using ClustalW alignment [52] implemented in MEGA version X [53] and were manually adjusted to allow maximum sequence
similarity. Bayesian inference (BI) was used to reconstruct the phylogenetic trees using
MrBayes version 3.2.7 [54] implemented in the CIPRES cluster (https://www.phylo.org/
portal2/home.action, accessed on 12 December 2021). The nucleotide substitution model
was determined by jModelTest v. 2.1.7 [55]. Following Drummond and Rambaut [56],
1,000,000 generations (four chains, four independent runs) were set up, and the analyses
were sampled every 1000 generations, with the first 25% of the samples discarded. Maximum likelihood analyses were conducted by the MEGA version X [53] using a TN93+G
substitution model based on 1000 bootstrap replicates.
4.5. Pathogenicity Test
Harvested mature mango fruit and leaves were washed under running water, immersed in 1.2% sodium hypochlorite solution for 2 min, rinsed twice with sterile distilled
water, and allowed to dry under a laminar flow hood. Thirty-seven isolates of Colletotrichum
were used for the pathogenicity test. Each Colletotrichum isolate was inoculated on unwounded fruit and leaves as described in Pongpisutta et al. [8]. Briefly, a mycelial plug from
the growing edge of 5-day-old PDA culture was placed onto the fruit and leaf surface of the
NDMST cultivar. A control treatment was performed using a non-colonized agar plug. The
inoculated samples were placed on trays lined with sterile moist paper towels and kept
in sealed plastic bags. Five days after inoculation at room temperature, evaluation of the
virulence was performed by measurement of lesion diameter (LD). This experiment was
performed in a completely randomized design (CRD) with ten replicates. One-way ANOVA
was performed using R software version 3.5.2 [57] with the agricolae package (Statistical
procedures for agricultural research) [58]. The means of the LDs were compared by the
least significant difference (LSD) test. Additionally, variation of disease lesion diameters
on NDMST mango fruit and leaves was compared amongst four Colletotrichum species by
using box plot analysis which was created in R software likewise.
5. Conclusions
A combination of multilocus phylogenetic analysis, phenotypic characters, and Koch’s
postulates, provides an effective strategy to overcome the problem of identification and
characterization of fungal species. We showed that molecular analysis of at least two
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loci (ITS and TUB2) provides accurate identification of Colletotrichum species causing
anthracnose disease in mango from central Thailand. This study represents the first report
that C. asianum and C. siamense were found to be causative agents of mango anthracnose
in Thailand.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/plants12051130/s1, Table S1: Morphological characteristics of
colonies, conidia, and appressoria of 37 mango Colletotrichum isolates from central Thailand. Table S2:
Colletotrichum isolates used in phylogenetic analysis, including all isolates in this study.
Author Contributions: Conceptualization, C.R. and R.P.; methodology, software, formal analysis,
writing—original draft preparation, C.R., P.K., S.B. and R.P.; supervision project administration,
funding acquisition, resource, S.C., C.R. and R.P.; investigation, data curation, C.R., P.K., S.B., V.P.,
S.C. and R.P.; validation, writing—review and editing, C.R., P.K., S.B., O.M., V.P., S.C. and R.P. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Postharvest Technology Innovation Center, Ministry of
Higher Education, Science, Research and Innovation, Bangkok, Thailand [Grant No. CRP.3. NOV11.8];
National Research Council of Thailand (NRCT) and Japan Society for the Promotion of Science (JSPS)
under the NRCT-JSPS joint research program (to R.P. and S.C.).
Data Availability Statement: Not applicable.
Acknowledgments: We thank Paul Morris and Raymond Larsen of Bowling Green State University
for critical reading of the manuscripts.
Conflicts of Interest: The authors declare no conflict of interest.
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