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Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia

Silmi Zhafarina, Arif Wibowo and Ani Widiastuti
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Background and Objective: Postharvest disease caused by Colletotrichum spp. caused major losses. The species of Colletotrichum are difficult to distinguish if only seen from their morphology. This study investigated Colletotrichum isolates associated with tropical fruits anthracnose using multi-genetic analysis and the cross-infection potency of each isolate among tropical fruits. Materials and Methods: The fruit samples were collected from markets in the Special Region of Yogyakarta, Indonesia and its surrounding area. The fruits affected by anthracnose subjected to isolation, resulting in 15 isolates. Morphology of colony and conidia then characterized and clustered with UPGMA. The seven representative isolates were selected for molecular identification. The multi-genetic analysis was used by combining ITS, Glyceraldehyde 3-phosphate dehydrogenase (gapdh) and tub2 sequence genes. A cross-infection test was conducted by using selected species from the multi-genetic analysis. Results: Multi-genetic analysis clustered the selected isolates into four species. Isolates from banana, avocado, papaya and citrus belonged to gloeosporioides species complex, including C. siamense, C. asianum and C. gloeosporioides. Isolates from apple, guava, mango and citrus belonged to acutatum species complex, including C. sloanei. The cross-infection test in this study showed that C. siamense could cause anthracnose on banana, apple, citrus and avocado, C. asianum on avocado, papaya, apple and citrus, C. gloeosporioides on citrus and apple, C. sloanei on apple, guava, citrus and papaya. Conclusion: The C. siamense, C. asianum, C. gloeosporioides and C. sloanei found associated with tropical fruits anthracnose. The potency of the cross-infection test revealed the board range in the pathogenicity of the Colletotrichum isolates.

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Silmi Zhafarina, Arif Wibowo and Ani Widiastuti, 2021. Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia. Pakistan Journal of Biological Sciences, 24: 53-65.

DOI: 10.3923/pjbs.2021.53.65

Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Fruits and vegetables are important commodities that affect the economy in developing countries such as Indonesia. The production of some popular tropical fruits has decreased by around 3% in a year. Postharvest product loss can reach 20-50% due to inappropriate handling of harvest and postharvest. In post-harvest, it is mostly caused by postharvest disease despite the use of modern storage facilities and technologies. Postharvest diseases reduce fruit quantity and quality, the fruits may not unsaleable but still reduce in value1-3. Well-known fungi caused major postharvest losses is Colletotrichum. Colletotrichum is the causal agent of anthracnose disease. It can affect many fruits such as chili, avocado, mango, banana, papaya, guava, citrus fruits, etc4.

Colletotrichum has many species and the taxonomic of the genus Colletotrichum changes frequently. Colletotrichum spp. are difficult to distinguish if they are only seen from their morphology. The difference is difficult to see because there are overlapping morphological characters in the Colletotrichum spp. The plasticity of Colletotrichum morphological characters make molecular technique analyzes more reliable for their classification. Identification of species using molecular techniques analysis is necessary. This helps early detection and provides appropriate guidance for the next steps in disease management5-7.

New reports of some novel Colletotrichum species in 2019 used multi-genetic analysis were described as C. javanense, C. makassarense and C. tainanense that associated with anthracnose of chili fruit in West Java (Indonesia), Makassar, South Sulawesi (Indonesia) and Tainan (Taiwan). Colletotrichum siamense and C. fructicola that included in 11 different Colletotrichum species identified in the same research was first reported causing anthracnose in chili in Indonesia, Sri Lanka, Thailand and Taiwan, although it has been reported as infecting many plant species before8.

Based on that information, cross-infection of Colletotrichum spp. can occur on fruits. Fruits that always available in the field and market and mostly cultivated in mixed-cropping systems make the potential for cross-infection cause greater susceptibility to anthracnose disease9. Cross-infection potential tests between fruits were conducted to provide information to develop an integrated system for controlling postharvest loss due to anthracnose disease10. Lakshmi et al.10 reported that C. gloeosporioides obtained from mango developed anthracnose symptoms on papaya, guava, acid lime, custard apple, pomegranate and cashew. The symptoms also developed on fruits when the isolates from six other fruits were inoculated on mango, with different susceptibility. Phoulivong et al.11 also reported the potential cross-infection of C. asianum, C. cordylinicola, C. fructicola, C. siamense and C. simmondsii to infect a wide host range. Suparman et al.9 reported C. gloeosporioides isolated from papaya and eggplant and C. capsici isolated from chili could infect all tested fruits (papaya, eggplant, chili) except common bean.

There is not much research studying the diversities of Colletotrichum species from tropical fruits and the relationship of each isolate, especially in Indonesia. Therefore, there is a need to renewing the data of Colletotrichum isolates from other tropical fruits with molecular data using multi-gene technologies, morphological character and pathogenicity to get updated information of the host-pathogen relationship. The purposes of this study were to investigate the phylogenetic relationship of Colletotrichum isolates associated with tropical fruits anthracnose and the cross-infection potency of each isolate among tropical fruits.


Study area: The study was carried out at the Laboratory of Plant Disease Clinic, Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Special Region of Yogyakarta, Indonesia from August, 2019 until April, 2020.

Fruits sampling: Fruits samples with anthracnose symptoms were collected from markets in the Special Region of Yogyakarta, Indonesia and its surrounding area. There were 7 fruits collected from markets, i.e., avocado, guava, banana, papaya, apple, mango and citrus.

Isolation of Colletotrichum spp.: Fruits with anthracnose symptoms were disinfected by using 70% alcohol by rubbing the surface. The area between the healthy and sick part was cut 1 cm×1 cm and cultured on Potato Dextrose Agar (PDA) medium on a Petri dish. The cultures were incubated for 4-7 days in room temperature. Isolates from various commodities were identified based on the morphology of Colletotrichum spp. following Weir et al.12 and Damm et al.13 Cultures of Colletotrichum sp. then were re-cultured in another Petri dish with PDA medium for subsequent tests.

Purification of Colletotrichum spp. with single-spore isolation: Suspension of Colletotrichum spp. was prepared by cutting 1×1 cm of 7 days culture on PDA medium and adding 500 μL sterile water in 1.5 mL tube. The suspension was homogenized by the vortex. The suspension obtained was scratched on a new PDA medium and incubated at room temperature for 12-24 hrs. The germinated conidium was taken under a microscope using a sterile needle and replaced to a new PDA medium. Incubation was conducted at room temperature.

Morphological analysis: Morphological characters were analyzed by characterizing the cultures include colony color, texture, conidiomata, growth rate, conidial dimension. The length and width of conidia were measured by choose 30 random conidia for each isolate after 10 days of incubation. Data of morphological character then was analyzed used NTsys 2.10 program and UPGMA method (Un-weighted Pair Groups with Arithmetical Averages). The Colletotrichum spp. isolates then were chosen as representatives according to their groups for further tests.

Molecular identification: Selected isolates represented each morphological group then were subjected to molecular identification. DNA of the isolates was extracted with Plant Mini Kit (Geneaid). The DNA obtained was amplified using the PCR technique using primers ITS1 and ITS414,15, primers tub2 (β-tubulin), T1 and T2216, primers gapdh (Glyceraldehyde 3-phosphate dehydrogenase), GDF1 and GDR117. The PCR was carried out in a total volume 25 μL, comprised of 9.5 μL miliQ sterile water, 12.5 μL DNA Taq polymerase (My Taq HS Red Mix; Bioline, London, United Kingdom), 1 μL forward primer, 1 μL reverse primer and 1 μL DNA template. Amplification was conducted using T100 Thermal Cycler (Biorad, California, United States). PCR program for ITS included an initial denaturation at 95°C for 5 min, followed by 35 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min and final cycle at 72°C for 5 min. The PCR program for gapdh and tub2 included an initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, 60°C for 45 sec, 72°C for 1 min and final cycle at 72°C for 10 min. PCR products were visualized with electrophoresis through 1% agarose gel at 50 V for 50 min and UV transilluminator with 1 kb DNA ladder (Thermo Fisher Scientific, Maltham, United States). DNA sequence analysis of the PCR product obtained was by sending it to the 1st Base, Malaysia.

Multi-gene phylogenetic analysis: The sequence of ITS, gapdh and tub2 genes of the chosen isolates were aligned by Clustal W to obtain a consensus sequence. Alignments were optimized manually in Bioedit. MEGA7.0. was used to build the phylogenetic tree with the maximum-likelihood (ML) algorithm. The phylogenetic tree was constructed with the combined ITS, gapdh and tub2 dataset. The sequences were compared with selected species (Table 1) and the NCBI sequence database was used the BLAST algorithm for approximate identification.

Cross-infection test: Selected species of Colletotrichum from multi-gene phylogenetic analysis then used for cross-infection tests. Host fruits were prepared by purchased healthy fruits which untreated, un-waxed, physiologically mature and unripe from the market. The surface of fruits was sterilized by rubbing the surface of the fruits using 70% alcohol. Sterilized fruits were placed in a plastic box with tissue paper then was sprayed with sterilized water to maintain at least 95% relative humidity. Fruits were inoculated using a wound and non-wound inoculation method.

The wound inoculation method was conducted by pin-pricked the fruits with a sterile needle in the middle portion of fruit, then mycelial disk from 10 days old Colletotrichum spp. was placed onto the wound. Non-wound inoculation method was without pin-pricked the same fruits, then mycelial disk from 10 days old Colletotrichum spp. was placed onto the surface of the fruits. The inoculated samples were incubated in the containers at 28-30°C in a 12 hrs light/dark cycle10.

Fruits used in cross-infection tests were guava (Psidium guajava), citrus (Citrus sp.), apple (Malus domestica), banana (Musa sp.), papaya (Carica papaya) and avocado (Persea americana) with seven treatments (isolates) and three replicates per fruit. The infection was measured based on lesion development on the symptom of fruit. Scored according to Montri et al.18:

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia

The data were analyzed used analysis of variance (p<0.05) with DMRT for multiple range tests using SAS V9.0.

Table 1: Isolates used for multi-genetic analysis in this study
GenBank accession number
Species Isolates name Host tub2 gapdh ITS
Acutatum species complex
C. abscissum COAD 1877 Citrus sinensis cv. Pera KP843135 KP843129 KP843126
C. acerbum CBS 128530 Malus domestica JQ950110 JQ948790 JQ948459
C. acutatum acutatum CBS 112996 Carica papaya JQ005860 JQ948677 JQ005776
C. citri CBS 134233 Citrus aurantifolia KC293661 KC293741 KC293581
C. fioriniae CBS 125396 Malus domestica JQ949950 JQ948629 JQ948299
IMI 324996 Malus pumila JQ949952 JQ948631 JQ948301
CBS 129938 Malus domestica JQ949947 JQ948626 JQ948296
CBS 129930 JQ949955 JQ948634 JQ948304
ATCC 28992 Malus domestica JQ949948 JQ948627 JQ948297
C. guajavae IMI 350839, CPC 18893 Psidium guajava JQ949921 JQ948600 JQ948270
C. limetticola CBS 114.14 Citrus aurantifolia JQ949844 JQ948523 JQ948193
C. paranaense CBS 134729 Malus domestica KC205060 KC205026 KC204992
C. paxtonii IMI 165753 Musa sp. JQ949936 JQ948615 JQ948285
C. sloanei BRIP48742 Litchi chinensis KU221365 KU221342 KU498289
C. simmondsii BRIP28519 Carica papaya FJ 907443 FJ 972580 FJ 972601
CBS294.67 Carica papaya FJ 907444 FJ 972581 FJ 972610
CBS 122122 Carica papaya JQ949927 JQ948606 JQ948276
Gloeosporioides species complex
C. aenigma ICMP 18608 Persea americana JX010389 JX010044 JX010244
C. alienum ICMP12071 Malus domestica JX010411 JX010028 JX010251
C. asianum IMI 313839, ICMP 18696 Mangifera indica JX010384 JX009915 JX010192
C. gloeosporioides CBS93597 Citrus sinensis FJ 907445 FJ 972582 FJ 972609
IMI 356878 Citrus sinensis JX010445 JX010056 JX010152
C. hystricis CBS 142411, CPC 28153 Citrus hystrix KY856532 KY856274 KY856450
C. kahawae subsp. ciggaro ICMP 12952 Persea americana JX010426 JX009971 JX010214
C. musae CBS 116870 Musa sp. HQ596280 JX010050 JX010146
C. psidii CBS145.29 Psidium sp. JX010443 JX009967 JX010219
C. queenslandicum ICMP1778 Carica papaya JX010414 JX009934 JX010276
C. siamense ICMP 12567 Persea americana JX010387 JX009940 JX010250
ICMP 17795 Malus domestica JX010393 JX010051 JX010162
C. tropicale CMM 4071 Mangifera indica KC517258 KC517181 KC329785
CMM 4243 Musa sp. KU213604 KU213601 KU213603
Dematium species complex
C. fructi CBS 346.37 Malus sylvestris GU228138 GU228236 GU227844
Boninense species complex
C. citricola CBS 134228 Citrus unshiu KC293656 KC293736 KC293576
CBS 134229 Citrus unshiu KC293657 KC293737 KC293577
CBS 134230 Citrus unshiu KC293658 KC293738 KC293578
C. constrictum CBS 128504, ICMP 12941 Citrus limon JQ005672 JQ005325 JQ005238
C. karstii CBS 113087 Malus sp. JQ005615 JQ005268 JQ005181
CBS 128524 Citrullus lanatus JQ005615 JQ005282 JQ005195
CBS 128551 Citrus sp. JQ005642 JQ005295 JQ005208
CBS 129832 Musa sp. JQ005611 JQ005264 JQ005177
CBS 129824 Musa AAA JQ005649 JQ005302 JQ005215
Unidentified species complex
C. musicola CBS 132885 Musa sp. MG601003 MG600798 MG600736
C. cliviae CMM 3742 Mangifera indica KC992327 KC702941 KC702980
C. plurivorum MAFF 305790 Musa sp. MG600993 MG600789 MG600726
C. tropicicola BCC 38877 Citrus maxima JN050246 JN050229 JN050240


Anthracnose symptoms on fruits sample: The symptoms of anthracnose disease associated with Colletotrichum spp. from varied tropical fruits in the Special Region of Yogyakarta market and its surrounding areas were slightly different on each fruit. The symptoms varied from brown to black spots (mango, banana) and light brown to dark lesion sunken areas (guava, citrus), some lesions had black or pink spore masses at the center part as it ages (apple, avocado, papaya) (Fig. 1).

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia
Fig. 1(a-g): Anthracnose symptoms in fruits caused by Colletotrichum spp.
(a) Guava, (b) Citrus, (c) Apple, (d) Avocado, (e) Mango, (f) Banana and (g) Papaya

Culture and conidia morphology: Total 15 Colletotrichum isolates were collected from varied tropical fruits. Colletotrichum isolates showed variations in cultural and morphological characteristics on PDA after 10 days of incubation under room temperature (Table 2). Most isolates were cottony and had the concentric ring. There was only one isolate that had a non-cottony texture and no concentric ring, it was APL-MLG collected from apple fruit. The color of the aerial view of all isolates was different such as brownish, greyish, white and greenish-grey. The color of the reverse view of all isolates was different such as brownish, greyish, white, pinkish and greenish-grey. The growth rate was also different among all isolates. PSG-JG isolate grew the fastest, APL-MLG isolate grew slower than all isolates.

Conidia produced by Colletotrichum isolates were fusiform and cylindrical with two ends acute or one end slightly obtuse (Table 2). There were only two isolates produced fusiform conidia, JRK-SMO and JRK-SMP isolate, collected from citrus fruits. The conidia size of all isolates were categorized by three groups, small for 8-11 μm conidia, medium for 11-14 μm conidia and big for conidia more than 14 μm conidia. APL-MLG, MGG-JG and JMB-GW4 isolate had small conidia. ALP-DMGO, JRK-SMO, JRK-KR5, JRK-KRM, PPY-GDN2 and PSG-JG isolate had medium conidia. ALP-DMGA, ALP-DRS, JRK-SMP, JRK-DRS, JRK-SL and MGG-SM2 isolate had big conidia.

Morphological character grouping was shown in Fig. 2, the first group had a small size of conidia and brownish mycelium color. The second group had a fast growth rate colony. The third group produced a white colony color and a fast growth rate. The fourth group was the only isolates that had a pinkish reverse side colony color. The fifth group produced greyish colony color, big size and cylindrical shape of conidia. The sixth group had a greenish-grey reverse side, medium-size and cylindrical shape of conidia and medium growth rate. The seventh group had the cylindrical shape of conidia, concentric ring and medium growth rate. APL-MLG, PSG-JG, JRK-SMO, PPY-GDN2, ALP-DMGA, JRK-KR5 and JMB-GW4 then were selected from each group as representative isolates. Figure 3 showed the aerial view, reverse view and conidia of 7 representative isolates of Colletotrichum spp. APL-MLG isolated from apple had brownish mycelium color {Fig. 3 (1, 8)} and small cylindrical conidia {Fig. 3 (15)}.

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia
Fig. 2: UPGMA dendrogram showing 7 groups of Colletotrichum spp. isolates
The dendrogram was built based on the similarity of the morphological character of Colletotrichum spp. isolates from tropical fruits. The coefficient of similarity of each group is above 75%

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia
Fig. 3(a-c):
Representative isolates of Colletorichum spp.
(a) Aerial view, (b) Reverse view and (c) Conidia Colletotrichum sloanei from (1,8,15) apple/APL-MLG, (2,9,16) Guava/JMB-GW4, C. gloeosporioides from (3,10,17) citrus/JRK-SMO, (4,11,18) citrus/JRK-KR5, C. asianum from (5,12,19) papaya/PPY-GDN2, (6,13,20) avocado/ALP-DMGA and C. siamense from (7,14,21) banana/PSG-JG

PSG-JG isolated from banana had a greyish colony color {Fig. 3 (7, 14)} and medium cylindrical conidia {Fig. 3 (21)}. JRK-SMO isolated from citrus had white mycelium color {Fig. 3 (3, 10)} and medium fusiform conidia {Fig. 3 (17)}. PPY-GDN2 isolated from papaya had a greyish aerial view, pinkish reverse view colony color{Fig. 3 (5, 12)} and medium cylindrical conidia {Fig. 3 (19)}. ALP-DMGA isolated from avocado had greyish mycelium color {Fig. 3 (6, 13)} and big cylindrical conidia {Fig. 3 (20)}. JRK-KR5 isolated from citrus had greenish-grey mycelium color {Fig. 3 (4, 11)} and medium cylindrical conidia {Fig. 3 (18)}. JMB-GW4 isolated from guava had greyish mycelium color, concentric ring {Fig. 3 (2, 9)} and small cylindrical conidia {Fig. 3 (16)}. The isolates then were subjected to molecular identification.

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia
Fig. 4(a-c):
PCR results for Colletotrichum spp. from varied tropical fruits
GeneRuler 1kb bp DNA ladder is shown in lane M, (a) Gapdh, (b) ITS and (c) Tub2 band position of all isolates from group representation (1,8,15) APL-MLG, (2,9,16) JRK-SMO, (3,10,17) ALP-DMGA, (4,11,18) JMB-GW-4, (5,12,19) PSG-JG, (6,13,20) PPY-GDN2 and (7,14,21) JRK-KR5

Molecular identification: Molecular identification of 7 representative isolates of Colletotrichum spp. performed using 3 primer pairs, i.e., ITS, gapdh and tub2. Figure 4 showed the PCR results of 7 representative isolates of Colletotrichum spp. (APL-MLG, JRK-SMO, ALP-DMGA, JMB-GW4, PSG-JG, PPY-GDN2, JRK-KR5). PCR amplified approximately 280 bp fragment from gapdh gene {Fig. 4A, (1-7)}, 550-600 bp fragment from ITS {Fig. 4B, (8-14)} and 750 bp from tub2 {Fig. 4C, (15-21)}. All selected isolates produced the expected amplicon sizes.

Multi-gene phylogenetic analysis: The phylogenetic tree was built by combining ITS, tub2 and gapdh sequence alignment of the selected isolates, BLAST algorithm and the reference species (Table 1). From 7 selected isolates, 5 isolates and 24 reference species were belong to gloeosporioides species complex, those 5 selected isolates were PSG-JG, PPY-GDN2, ALP-DMGA. JRK-KR5 and JRK-SMO (Fig. 5a). The other 2 selected isolates and 32 reference species belonged to acutatum species complex, those 2 isolates were APL-MLG and JMB-GW4 (Fig. 5a).

The analysis of gloeosporioides species complex using ITS, gapdh and tub2 sequence alignment comprised of 29 isolates with C. fructi CBS 346.37 (Fig. 5b) as the outgroup species clustered citrus isolates with C. gloeosporioides, papaya and avocado isolate with C. asianum and banana isolates with C. siamense. The analysis of acutatum species complex comprised of 34 isolates (Fig. 5c) with C. gloeosporioides ICMP 17821 as outgroup species clustered the apple and guava isolate with C. sloanei.

Cross-infection test: The percentage of fruits length in the cross-infection test of representative Colletotrichum isolates on tropical fruits, some isolates could infect non-host fruits but in different pathogenicity (Table 3). On wound inoculation, Colletotrichum sloanei from apple infected guava, citrus and papaya as non-host fruits with the percentage of infections 23.11, 6.19 and 3.62%, respectively. Colletotrichum sloanei from guava infected apple and citrus as non-host fruits with the percentage of infection 11.33 and 9.44%, respectively. Colletotrichum siamense from banana infected apple, citrus and avocado as non-host fruits with the percentage of infection 2.00, 9.74 and 8.33%, respectively. Colletotrichum gloeosporioides from Citrus reticulata (JRK-SMO) infected apple (6.67%) whereas isolates from Citrus sinensis (JRK-KR5) could not infect another fruit except the original host in the same genus, which was Citrus reticulata. Colletotrichum asianum from avocado infected apple (1.21%) and citrus (10.00%), whereas the same species isolated from papaya only infected citrus as non-host fruits with the percentage of infection 8.89%. On non-wound inoculation in this study, there were three isolates infected the original host, C. sloanei from guava (2.92%), C. asianum from papaya (2.98%) and avocado (1.00%). One isolates had the potency of cross-infection to other host, C. sloanei from guava infected apple as non-host fruit with the percentage of infection 5.33%. Table 4 showed a summary of potential cross-infection of representative Colletotrichum isolates among tropical fruits in this study, some isolates of Colletotrichum infected the other host and original host from they were isolated.

Table 2: Colony and conidia characteristic of each isolates Colletotrichum spp. from varied tropical fruits in Special Region of Yogyakarta
Colony color Conidia size (μm)
  Growth rate
Isolate name Aerial view Reverse view Colony texture Concentric ring Conidiomata Black spot Conidial shape Length Width (mm day1)
APL-MLG Brownish Brownish Non-cottony - - - Cylindrical 8.40±4.06 4.02±1.12 5.68
ALP-DMGA Greyish Greyish Cottony v v v Cylindrical 15.17±1.47 3.41±0.87 10.75
ALP-DMGO White orange White orange Cottony v v - Cylindrical 12.13±4.59 3.50±1.55 11.15
ALP-DRS White greyish Greyish Cottony v - v Cylindrical 16.16±4.99 2.99±0.65 11.28
JRK-SMO White White Cottony v v v Fusiform 12.74±1.36 4.56±0.97 11.87
JRK-SMP White White Cottony v v v Fusiform 14.66±1.64 4.42±0.91 11.45
JRK-DRS Greyish Greyish Cottony v v v Cylindrical 14.87±1.53 4.78±0.67 11.37
JMB-GW4 White greyish Greyish Cottony v - - Cylindrical 9.55±2.74 3.67±0.72 7.12
JRK-KR5 White greyish Greenish grey Cottony v v - Cylindrical 11.40±1.47 3.58±0.88 8.20
JRK-KRM Greenish grey Greenish grey Cottony v v - Cylindrical 12.36±1.78 3.43±0.68 9.67
JRK-SL Greyish Greyish Cottony v - - Cylindrical 15.41±2.92 3.76±0.91 8.67
MGG-JG Brownish grey Brownish Cottony v - - Cylindrical 8.38±2.48 4.39±1.07 10.08
MGG-SM2 White White Cottony v - - Cylindrical 14.19±2.15 3.43±0.69 9.67
PPY-GDN2 Greyish Pinkish Cottony v v v Cylindrical 13.65±3.66 3.44±0.79 11.32
PSG-JG White greyish Greyish Cottony v v - Cylindrical 13.78±1.91 4.67±0.92 12.57
*v: Isolate had the characteristic of morphology, -: Isolate did not have the characteristic of morphology

Table 3: Percentage of fruits length infected in cross-infection test of Colletotrichum species on tropical fruits
Fruit length infected (%)
Apple Guava Banana Citrus Papaya Avocado
Colletotrichum species Isolates name Host w nw w nw w nw w nw w nw w nw
C. sloanei APL-MLG Apple 3.64a 0.00a 23.11a 0.00a 0.00b 0.00a 6.19a 0.00a 3.62a 0.00a 0.00a 0.00a
JMB-GW4 Guava 11.33a 5.33a 20.00b 2.92a 0.00b 0.00a 9.44a 0.00a 0.00a 0.00a 0.00a 0.00a
C. siamense PSG-JG Banana 2.00a 0.00a 0.00c 0.00a 9.33a 0.00a 9.74a 0.00a 0.00a 0.00a 8.33a 0.00a
C. gloeosporioides JRK-SMO Citrus 6.67a 0.00a 0.00c 0.00a 0.00b 0.00a 5.56a 0.00a 0.00a 0.00a 0.00a 0.00a
JRK-KR5 Citrus 0.00a 0.00a 0.00c 0.00a 0.00b 0.00a 15.00a 0.00a 0.00a 0.00a 0.00a 0.00a
C. asianum PPY-GDN2 Papaya 0.00a 0.00a 0.00c 0.00a 0.00b 0.00a 8.89a 0.00a 2.98a 2.98a 0.00a 0.00a
ALP-DMGA Avocado 1.21a 0.00a 0.00c 0.00a 0.00b 0.00a 10.00a 0.00a 0.00a 0.00a 9.00a 1.00a
*Means with the same letter in each column are not significantly different from each other based on DMRT, w: Wound, nw: Non-wound

Image for - Multi-genetic Analysis of Colletotrichum spp. Associated with Postharvest Disease of Fruits Anthracnose in Special Region of Yogyakarta, Indonesia
Fig. 5(a-c):
Phylogenetic analysis using Maximum Likelihood (ML) algorithm combined gene of ITS, gapdh and tub2
(a) Sequence alignment showing the separation of Colletotrichum isolates into gloeosporioides species complex, boninense species complex, dematium species complex and acutatum species complex, the four clades containing tropical fruits isolates are indicated by blocks, (b) The analysis of gloeosporioides species complex was used C. fructi as outgroup species and (c) The analysis of acutatum species complex was used C. gloeosporioides as outgroup species

Table 4: Cross infection potency of Colletotrichum species among tropical fruits
Cross infection potency
Colletotrichum species Isolates name Host
C. sloanei APL-MLG Apple
JMB-GW4 Guava
C. siamense PSG-JG Banana
C. gloeosporioides JRK-SMO Citrus
JRK-KR5 Citrus
C. asianum PPY-GDN2 Papaya
ALP-DMGA Avocado
*+: Fruits infected by Colletotrichum isolates, -: Fruits not infected by Colletotrichum isolates


The results indicated that Colletotrichum species isolated from tropical fruits in the Special Region of Yogyakarta, apple, banana, citrus, avocado, papaya and guava isolates showed variations in cultural and morphological characters (Fig. 3). The morphological features including the cultural characteristics, size and shape of the conidia, present of conidiomata and colony growth rate than were investigated into the phylogenetic analysis (Table 2). This approach was explained by Cai et al.5 that morphological characteristics and molecular data were needed to be linked as a polyphasic approach.

The C. gloeosporioides had a large range of colony color and growth rate, also very common on Citrus sp.12. Both isolate from citrus (JRK-SMO and JRK-KR5) had a similar colony color of C. gloeosporioides but different in size and shape of conidia compared with the description from Weir et al.12, Prihastuti et al.19, Aiello et al.20 and Ramos et al.21. The other isolates from the same group in UPGMA dendrogram (Fig. 2), JRK-SMP, ALP-DMGO and JRK-KRM, had the characterization that similar to C. gloeosporioides. Fusiform conidia commonly found in C. acutatum, but two of the isolates (JRK-SMO and JRK-SMP) of C. gloeosporioides in this study were found to have a fusiform conidia {Fig. 3 (17)}.

The isolates derived from citrus (JRK-SMO, JRK-SMP, JRK-KR5, JRK-KRM) in this study were similar to C. gloeosporioides described by Ramos et al.21 and Rhaim and Taylor22 who conducted the same study using multi-gene analysis in the detection of Colletotrichum in citrus fruits. Obtained C. gloeosporioides isolates also had various colony colors and conidial sizes. Moges et al.23 also explained that C. gloeosporioides was a common species associated with anthracnose in citrus. Avocado isolate (ALP-DMGO) which was in a group with group 3 (Fig. 2), had morphological characteristics similar to those explained by Sharma et al.24, C. gloeosporioides obtained from avocados had orange colony colors and cylindrical conidial shapes and measuring between 12.0-17.0×3.5-6.5 μm.

Isolate from banana (PSG-JG) clustered with C. siamense YN40-1-2 isolated from mango fruits had the same colony color and shape of conidia25. A study by Uysal and Kurt26 explained the morphological character of C. siamense causing anthracnose on banana fruits had white to grey colony color and concentric ring, these were similar to the isolates from this study, but the conidia shape was different. C. siamense previously reported associated with coffee berries21, mango and avocado27.

Isolate from papaya (PPY-GDN2) and avocado (ALP-DMGA) clustered with C. asianum VN4-2 and C. asianum ICMP 18696 isolated from mango fruits and had the same colony color, the shape of conidia, also in the range of conidia size and mycelium growth rate28. Isolates from the same group in UPGMA dendrogram, JRK-DRS and ALP-DRS also had the same morphology character as C. asianum. C. asianum commonly found from mango, but Giblin et al.27 study found one isolate of C. asianum isolated from mango was pathogenic to avocado. Phoulivoung et al.29 studied the Colletotrichum species associated with tropical fruits found that C. asianum was obtained from mangoes, whereas the Colletotrichum species obtained from papaya was C. fructicola which also in the gloeosporioides species complex. Previous studies conducted by Prihastuti et al.21 reported that C. asianum was one of Colletotrichum species associated with coffee berries in northern Thailand.

Apple isolates (APL-MLG) and guava isolate (JMB-GW4) clustered with C. sloanei BRIP 48742. The conidia size and shape were quite similar compared to Damm et al.13. Isolates from mango (MGG-JG, MGG-SM2) and isolate from citrus (JRK-SL) and the other isolates from the same group in UPGMA dendrogram also had a similar morphological characterization with C. sloanei. Conidia of the representative Colletotrichum isolates were cylindrical with one end round and one end acute {Fig. 3 (15,16)}. This is the first report of C. sloanei on apple, guava, mango and citrus. The host range and pathogenicity of C. sloanei is still little known. C. sloanei belongs to acutatum species complex and best distinguished from other species in acutatum species complex with gapdh or tub sequences30.

In this study, colony and conidia morphology alone could not be distinguished between Colletotrichum subspecies, nor did they differentiate between acutatum and gloeosporioides species complex. Therefore to identify Colletotrichum to species level, molecular analysis was needed, especially with multi-genetic analysis. Previous studies have suggested that acutatum species complex and gloeosporioides species complex were the most confusing species complex to distinguish from their morphological parameters12,13. It was also mentioned that Colletotrichum species had very large conidia size range so that overlap could occur in morphological observations. Colletotrichum species from different hosts would form different colonies and conidia, apart from the host this could also be influenced by environmental conditions of isolate growth (growing media, temperature, light, etc.)12,31,32.

Cross-infection test results in this study showed that there was the potency of cross-infection among tropical fruits. Isolates evaluated were able to produce symptoms in their original hosts, but not always on another (Table 4). Previous studies10,33 reported similar results but with different isolates. Colletotrichum sloanei from guava had the highest percentage of infection on apple (11.33%), although it was not statistically different (Table 3) and interestingly the isolate of C. sloanei from apple had the highest percentage of infection on guava fruits (23.11%). Lakshmi et al.10 also reported that the isolate of Colletotrichum could be more aggressive to other tested fruits than the original host. This was different from Hayden et al.31 previously reported isolates of Colletotrichum were more aggressive in infecting the host from which they were originally isolated.

All isolates in this study could infect citrus fruits, but isolate C. gloeosporioides from citrus could not infect all non-host fruits. JRK-KR5 isolate only found infected citrus, this could be the sign this isolate is host-specific. Phoulivong et al.11 cross-infection study showed that Colletotrichum strains could infect more than one host and one host also could be infected with many Colletotrichum species. The same species isolated from different hosts had different cross-infection ability and this should be considered when establishing new species. The different capabilities of Colletotrichum spp. to infect various kinds of fruits could be attributed to variations in the compositions of each kind of fruits10. Fruits susceptibility to Colletotrichum infection was connected to the degree of antifungal inhibitor present in these fruits34.

Many isolates also caused lesions on fruits in wounded inoculation but not in unwounded inoculations. This has also happened in the previous study. This situation related to quiescent infection of the species, which was the lifestyle of Colletotrichum spp., where infection occurred at un-ripen fruit then developing fruit rot as the fruit ripens or the condition and environmental was supportive6,35.

In a previous study, C. sloanei was reported to cause anthracnose on Litchi chinensis and the host range was little known12,30. Colletotrichum siamense was also previously reported caused anthracnose on peach, pear, coffee berries, citrus, guava, mango, chili, papaya11,21,35-37. Colletotrichum gloeosporioides previously was reported to associate with citrus, pear, avocado, mango, dragon fruit, olive, papaya35,38-41. Colletotrichum asianum previously was reported to associate with coffee berries, mango, chili, rose apple11,21,42-45.

The current finding in this study showed Colletotrichum species were obtained from apple, citrus, guava, banana, mango, avocado and papaya. Cross-infection test results showed different pathogenicity of each isolate. This data can be used to see host-pathogen interactions of Colletotrichum. However, it is still necessary to identify the species complex of Colletotrichum and study the potency of cross-infection on different tropical fruits to add the data for this pathogen.


Multi-genetic analysis and morphological identification in this study revealed C. siamense, C. asianum, C. gloeosporioides and C. sloanei associated with tropical fruits anthracnose in Special Region of Yogyakarta, Indonesia. According to the author’s knowledge, this is the first report of C. sloanei on apple, guava, mango and citrus in Indonesia. All isolates in this study had the potency of cross-infection, although each isolate varied in degrees of pathogenicity.


This study discovered the Colletotrichum species associated with tropical fruits anthracnose by using multi-gene analysis and the cross-infection potency of the species that can be beneficial for the management of anthracnose caused by Colletotrichum on tropical fruits. This study will help the researchers to uncover the critical areas of the identification species complex of Colletotrichum in the areas that many researchers were not able to explore. Thus, a new theory on identifying species of Colletotrichum may be arrived at.


The authors are deeply gratitude this research was fully sponsored by internal funding of Plant Disease Laboratory of the Faculty of Agriculture, Universitas Gadjah Mada, Indonesia (Grant No. 2/LAB-IPT/UGM/2019).


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