Identification and Characterization Colletotrichum spp. Causing Mango Dieback in Indonesia

Article information

Plant Pathol J. 2024;40(4):399-407
Publication date (electronic) : 2024 August 1
doi : https://doi.org/10.5423/PPJ.NT.04.2024.0061
1Graduate Student of Master Program in Phytopathology, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
2Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
*Corresponding author. Phone) +62-822-31775737, FAX) +62-822-31775737, E-mail) arif_wibowo@ugm.ac.id
Handling Editor: Wonsu Cheon
Received 2024 April 9; Revised 2024 June 6; Accepted 2024 June 10.

Abstract

Dieback disease in mango trees has been observed in Indonesia, particularly in Java Island, with the causal agent remaining unidentified. One of the important pathogens that are responsible for causing mango dieback is Colletotrichum. Field surveys were conducted in various mango cultivating areas in Java Island, Indonesia to assess prevalence of Colletotrichum as dieback disease pathogen. Eleven Colletotrichum isolates were recovered from symptomatic dieback twigs and morphologically characterized. Genetic diversity fingerprint analysis was carried out using rep-PCR. Phylogenetic analysis identified isolates as belonging to Colletotrichum asianum and Colletotrichum cairnsense using partial sequences of four gene regions, including ITS, ACT, GAPDH, and TUB2. Pathogenicity tests on mango seedlings cv. Arumanis showed that all fungal isolates were responsible for causing dieback symptoms. Subsequently, symptomatic tissue was reisolated to fulfill Koch’s Postulate. This study represented new funding for two species of Colletotrichum causing mango dieback in Indonesia.

Mango dieback is a significant disease that is affected by several fungal pathogens, including Botryosphaeria dothidea, Diplodia sp., and Ceratocystis sp. (Saeed et al., 2017). According to da Silva et al. (2022), the fungal pathogen that causes mango dieback belongs to the Botryosphaeriaceae family and Fusarium genus. Savant and Raut (2000) reported that dieback of mango stone grafts might be attributed to Colletotrichum gloeosporioides Penz. and Botryodiplodia theobromae Pat. The dieback symptoms infected by C. gloeosporioides included the development of dark to brown circular or irregular spots of leaves resulting in elongated black necrotic areas. Also, the development of acervuli was observed on infected shoots or tips of the twig. Additionally, recent discoveries by Hassan et al. (2022) have identified C. gloeosporioides to be fungal pathogen associated with dieback disease in mangoes, as unveiled in a metagenomic nanopore sequencing study.

Colletotrichum is widely recognized for inducing mango disease with potential yield losses of up to 100% (Li et al., 2019; Sharma and Shenoy, 2016). In Indonesia, mango dieback cases linked to Colletotrichum infection have been particularly unreported where reviews have predominantly focused on mango fruit anthracnose disease particularly in Java Island, as the mango production center (Benatar et al., 2020). Despite the Colletotrichum affecting various plant parts, significant yield losses still manifest during the fruit-ripening stage when anthracnose symptoms become apparent (Widiastuti et al., 2023). This study aims to identify and characterize Colletotrichum spp. causing mango dieback in Indonesia which needs further concern for biosecurity.

For this study, symptomatic dieback twig samples were purposefully collected from various mango production centers in Java, Indonesia (Fig. 1). The sample had necrosis on twigs, petioles, and leaves, and the necrotic lesion initiated with irregular blackish-brown discoloration, expanding to cause dried areas (Fig. 2A–C).

Fig. 1

Survey area of mango twig dieback in Java Island, Indonesia. The colored map was districts where the collection sample location was arranged.

Fig. 2

Dieback symptoms on mango tissue caused by Colletotrichum manifested as necrosis on branches, twigs, petioles, and leaves (A–C). The initial presentation included small, angular, brown-to-black specks that could expand into dried and lifeless areas. Arrows indicate the symptoms of dieback caused by Colletotrichum.

Infected twig pieces were subjected to disinfection with 70% ethanol for 1 min, followed by 1% sodium hypochlorite (NaOCl) for 1 min. They were then rinsed twice with sterile ddH2O and dried using sterile filter paper. Two pieces were placed into potato dextrose agar (PDA) medium with 5 μl lactic acid, and the mixture was incubated at 28°C for 3 days. The resulting colonies, representing a single conidium of Colletotrichum, were recultured and incubated under the same conditions (Choi et al., 1999). In this study, 11 Colletotrichum spp. isolates were obtained from different cultivars, consisting of Manalagi, Arumanis, Gadung, Golek, and Gedong Gincu (Table 1).

Colletotrichum spp. pure isolates used in this study

Genomic DNA was extracted from pure mycelia of Colletotrichum isolates grown in PDA at room temperature 25°C for 7 days using the Taiwan Geneaid Genomic DNA Mini Kit (Plant) (Geneaid Biotech Ltd., New Taipei, Taiwan), following the manufacturer’s protocol. The genetic diversity fingerprints of Colletotrichum spp. were assessed through rep-PCR (repetitive element sequence-based PCR), using two primer sets, including BOX and ERIC. The PCR amplification and visualization method is based on Pramunadipta et al. (2022). PCR mixture consisted of 12.5 μl (dNTPs; MgCl2; 2× MyTaq HS Red Mix, Bioline, Meridian Bioscience, Cincinnati, OH, USA), 1 μl for each primer (100 μM), 2 μl template DNA isolates, and 8.5 μl ultrapure water, resulting in a total volume of 25 μl. Rep-PCR using BOX (5′-CTACGGCAAGGCGACGCTGACG-3′), ERIC1 (5′-ATGTAAGCTCCTGGGGATTCAC-3′), and ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) was conducted with the annealing temperatures set to 53°C for BOX and 52°C for ERIC. Polymorphic bands, observed between 100 and 3,000 base pairs, were shown in Fig. 3. A dendrogram was constructed using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis on rep-PCR based on the presence bands amplified with BOX and ERIC primers in Ntsys 2.2 software. Several isolates represented each banding pattern identical group, and 11 isolates were divided into four major groups based on a similarity coefficient of 70% (Fig. 4). The first group, containing only one fungal, was distinct from others (groups 2, 3, and 4), suggesting that this fungal isolate belonged to different clades of Colletotrichum species complex. The representative isolates from each group were chosen for molecular identification. Notably, for the 2nd and 4th groups, multiple isolates were selected to represent the origin of sample accurately. MLG-TJB, HM-IJB, HM-BDIY, MLG-TJT, MLG-PAJT, and HM-PAJT were selected for phylogenetic analysis using partial sequences of four gene regions (internal transcribed spacer [ITS], partial actin [ACT], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and β-tubulin [TUB2]). Patricia et al. (2021) mentioned that the absence and presence of band information were used to build UPGMA tree to compare the amplification profile of Colletotrichum strains. Genetic variability accessed by rep-PCR analysis constructed into a dendrogram phylogenetic also supports the genetic differences among the Colletotrichum isolates.

Fig. 3

DNA fingerprints of Colletotrichum spp. causing dieback on mango tree generated with primer sets BOX (A) and ERIC (B) for Colletotrichum spp. Lane 1, MLG-TJB; lane 2, HM-IJB; lane 3, GG-IJB; lane 4, MLG-IJB; lane 5, MLG-TJT; lane 6, HM-BDIY; lane 7, HM-SDIY; lane 8, HM-PAJT; lane 9, GAD-PAJT; lane 10, MLG-PAJT; lane 11, GO-PAJT; left M, 100 bp ladder Geneaid; right M, 1 kb ladder Geneaid.

Fig. 4

Dendrogram resulting from Unweighted Pair Group Method with Arithmetic Mean cluster analysis of DNA banding profiles generated with primer sets combined of BOX and ERIC for Colletotrichum spp. causing dieback on mango. The isolates in the red sign were selected for phylogenetic analysis.

The ITS region, TUB2, ACT, and GAPDH genes, were amplified using set primers of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), BTUB2fd (5′-GTBCACCTYCARACCGGYCARTG-3′) and BTUB4rd (5′-CCRGAYTGRCCRAARACRAAGTTGTC-3′), ACT512F (5′-ATGTGCAAGGCCGGTTTCGC-3′) and ACT783R (5′-TACGAGTCCTTCTGGCCCAT-3′), GDF1 (5′-GCCGTCAACGACCCCTTCATTGA-3′) and GDR1 (5′-GGGTGGAGTCGTACTTGAGCATGT-3′). PCR amplification and visualization adjustment were performed based on Wang et al. (2021), with annealing temperatures were set to 52°C, 58°C, 60°C, and 55°C for ITS, ACT, GAPDH, and TUB2, respectively. The results of PCR amplification on six isolates showed that all isolates amplified in the range of 600 bp for the ITS and 500 bp for the target β-tubulin region. Meanwhile, the target areas of the Actin and GAPDH were amplified in the range of 300 bp. The phylogenetic tree was constructed from concatenated ITS, ACT, GAPDH, and TUB2 sequences of Colletotrichum spp. samples and reference Colletotrichum isolates from GenBank (Table 2) using the maximum likelihood method with the Kimura 2-parameter model, and it was tested by 1,000 bootstrap replications.

Colletotrichum spp. ex-type used in phylogenetic analysis

The phylogenetic tree (Fig. 5) showed that six representative isolates formed two main groups with a high bootstrap value exceeding 50%. HM-BDIY, MLG-PAJT, and HM-PAJT had close relations to Colletotrichum asianum CMM4067, while HM-IJB and MLG-TJT clustered with C. asianum CMM4056. On the other hand, MLG-TJB was separated and clustered with Colletotrichum cairnsense AC11. It was observed that C. asianum belonged to Colletotrichum gloeosporioides species complex, whereas C. cairnsense belonged to Colletotrichum acutatum species complex (de Silva et al., 2017). The identification was in line with the genetic fingerprint results obtained from rep-PCR. The sequences of the six isolates (24 sequences in total) have been deposited in GenBank with the following accession numbers, ITS: OR945809-OR945814, actin: PP091732 to PP091736 and PP091747, GAPDH: PP091737 to PP0091741 and PP091748, TUB2: PP091742 to PP091746 and PP091749.

Fig. 5

The phylogenetic tree of six representative isolates from this study was built using concatenated sequences of the internal transcribed spacer (ITS), partial actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) genes. Bootstrap support values from maximum likelihood above 50% were shown at each node. Monilochaetes infuscans CBS869.96 was used as the outgroup.

Macroscopic and microscopic morphological observations were conducted on 10-day-old colonies. In general, Colletotrichum isolates produced light to dark olive-grey mycelium, with the center of colonies covered by orange conidia masses (Uysal et al., 2022). This study recorded a mycelial growth rate of 7.24 mm/d to 7.89 mm/d, as shown in Table 3. C. asianum had cottony mycelium, predominantly white, occasionally white grayish with sparse aerial hyphae (Fig. 6A). In this study, isolates identified as C. asianum exhibited morphological characteristics that closely matched those described by Benatar et al. (2020). The authors used the morphology of the MLG-TJT to represent C. asianum isolates in this figure, as they were the closest to the reference Meanwhile, C. cairnsense had white-greenish cottony mycelium (Fig. 6F). De Silva et al. (2017) also mentioned that C. cairnsense had a pale white-grey to olivaceous grey mycelium with a whitish margin. The reverse side of both species was white, typically forming concentric rings and dark spots as mature conidiomata developed (Fig. 6B and G). Additionally, both species produced masses of conidia (conidiomata) as presented in Fig. 6C and H.

Characteristics of conidia and mycelial growth

Fig. 6

Morphological characters of Colletotrichum asianum (isolate MLG-TJT) (A–E) and C. cairnsense (Isolate MLG-TJB) (F–J). (A, B, F, G) Colony of aerial and reverse on potato dextrose agar, 10 days growth from the single culture of conidia. (C, H) Conidiomata. (D, I) Conidia. (E, J) Appressoria and setae (setae only observed on C. asianum). Scale bars = 10 μm.

Microscopic characterization was observed using an Olympus CX31 microscope (Tokyo, Japan) and Miconos Optilab (PT Miconos, Yogyakarta, Indonesia) and it showed that C. asianum and C. cairnsense produced hyaline, smooth-walled cylindrical conidia with distinguishing features. For instance, C. asianum had obtuse ends, and the conidia of C. cairnsense had acutely rounded ends (Fig. 6D and I). The length and width of the conidia were measured by randomly selecting 50 conidia using a calibrated ImageRaster software (Ramos et al., 2016). Conidia measured 7–18.6 μm in length, 2.3–6.9 μm in width, and had a length/width ratio of 2.56–3.23 (Table 3). The formation of appressoria was observed using slide culture technique by growing the isolates on PDA (Siddiquee, 2017). Both C. asianum and C. cairsense formed appressoria originating from mycelia, characterized by dark brown, ovoid to irregular structures found at the ends of hyphae. According to Prihastuti et al. (2009), in slide culture, C. asianum formed appressoria that were brown to dark brown, ovoid, clavate to irregular in shape and often became complex with age. In contrast, de Silva et al. (2017) reported that C. cairnsense formed appressoria single that was medium brown, smooth-walled, subglobose, ovoid to ellipsoidal. In this study, only C. asianum produced setae that were perpendicular in shape and dark brown (Fig. 6E and J). C. asianum was initially reported in Indonesia by Benatar et al. (2020) in connection with mango anthracnose in Indramayu. On the other hand, C. cairsense has not been previously recorded in the country but was first identified in Australia. C. cairnsense sp. nov. was recognized as the causative agent of chili anthracnose by de Silva et al. (2017). According to the morphology observed in this study. The conidia of C. gloeosporioides species complex were cylindrical with obtuse ends, while C. acutatum species complex had cylindrical conidia with two acute ends or one end slightly obtuse.

Pathogenicity test was conducted on 8-month-old mango seedlings, specifically cv. Arumanis, with an average height of approximately 50 cm per seedling. cv. Arumanis were selected for this pathogenicity test because these cultivars are widely cultivated in Indonesia and frequently show dieback symptoms due to Colletotrichum infection, despite the lack of official reports documenting these cases. The test was performed exclusively on representative isolates identified through phylogenetic analysis, and all isolates of both C. asianum and C. cairnsense showed pathogenicity (Fig. 7B and D). Moreover, inoculation followed the method outlined by Mayorquin et al. (2019) with modifications. An active hyphal plug (approximately 5 mm in diameter) from a 14-day PDA culture of Colletotrichum spp. was placed on the twig tip, creating wounds up to 3 mm in diameter and 2 mm in depth, and covered with a moist sterile tissue then sealed. Control treatments were carried out by placing a sterile plug on the seedling with identical wounds. This process was repeated four times and incubated at greenhouse temperatures (approximately 28°C). A necrotic lesion was observed behind dieback lesion (Fig. 7C and E), with an orange-to-black conidial mass visible on the necrotic area (Fig. 7D). The control twig showed no symptoms (Fig. 7A), and the virulence assessment measured the lesion area on the 20th day after inoculation. All six representative isolates had virulence, with TJT-MLG (188 mm2) and PAJT-HM (119 mm2) identified as the most aggressive isolates (Fig. 7G).

Fig. 7

Pathogenicity test of six isolates representative of Colletotrichum spp. causing dieback symptom on mango. (A) Control twig. (B, C) Dieback necrosis with a blackish lesion by cross-section on the tip of twig inoculated by C. asianum. (D, E) Dieback necrosis with a blackish lesion by cross-section lesion on the tip of twig inoculated by C. cairnsense. (F) Orange conidial mass captured by Lumix G Mirrorless (DSLM) Cameras DC-GH6 (Kadoma, Osaka, Japan). The arrows of A, B, and D indicate symptoms that appear at the point of inoculation. (G) Lesion area was measured in four perpendicular ways and means followed with the same letter are not significantly different on 95% confidence interval (P > 0.05) analyzed with Tukey honestly significant difference test.

This study found that C. asianum was in line with several observations, indicating the isolates were considered the most significant fungal associated with mango diseases. According to Benatar et al. (2020), C. asianum was a virulent pathogen causing mango anthracnose in Indramayu, Indonesia. Vitale et al. (2020) also mentioned that C. asianum was a common species responsible for mango anthracnose, reported in various countries such as Australia, Brazil, China, Ghana, Japan, Malaysia, Mexico, Panama, Philippines, South Africa, Sri Lanka, Thailand, and Florida. Other species in C. acutatum species complex, particularly C. cairnsense, were newly recognized causes of mango diseases, despite previously being identified to be pathogens on Chili in Australia.

In conclusion, dieback studies associated with Colletotrichum infection were significantly scarce, but this study provided a new examination of mango dieback in Indonesia. It represented the new documentation of C. asianum and C. cairnsense as the causative pathogens. Further investigations were required to explore and quantify the occurrence and intensity of mango dieback caused by Colletotrichum in Indonesia. Accuracy in diagnosing causal pathogens was crucial for the efficacy of disease management.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This study has been funded by the research grant of the Indonesian Endowment Fund for Education (LPDP)-Ministry of Finance, Indonesia. We thank Dr. Adyatma Wirawan Santosa, S.P., M.Sc. for technical support in phylogenetic tree analysis and Sri Giyanti for technical support in the laboratory. This report is a part of master thesis research conducted by the first author, under supervision corresponding and third author.

References

Benatar G. V., Wibowo A., Suryanti . 2020;First report of Colletotrichum asianum associated with mango fruit anthracnose in Indonesia. Crop Prot. 141:105432.
Choi Y.-W., Hyde K. D., Ho W. W. H.. 1999;Single spore isolation of fungi. Fungal Divers. 3:29–38.
da Silva L. A. B., Leite D. M., Capucho A. S.. 2022;Chemical control of dieback and mango malformation in a semiarid region. Agric. Parasitol. 89:e00542020.
de Silva D. D., Ades P. K., Crous P. W., Taylor P. W. J.. 2017; Colletotrichum species associated with chili anthracnose in Australia. Plant Pathol. 66:254–267.
Hassan A. H., Bebawy A. S., Saad M. T., Mosaad G. S., Saad B. T., Eltayeb W. N., Aboshanab K. M.. 2022;Metagenomic nanopore sequencing versus conventional diagnosis for identification of the dieback pathogens of mango trees. BioTechniques 73:261–272.
Li Q., Bu J., Juan Shu, Yu Z., Tang L., Huang S., Guo T., Mo J., Luo S., Solangi G. S., Hsiang T.. 2019; Colletotrichum species associated with mango in southern China. Sci. Rep. 9:18891.
Mayorquin J. S., Nouri M. T., Peacock B. B., Trouillas F. P., Douhan G. W., Kallsen C., Eskalen A.. 2019;Identification, pathogenicity, and spore trapping of Colletotrichum karstii associated with twig and shoot dieback in California. Plant Dis. 103:1464–1473.
Patricia L.-C. C., del Socorro R.-G. M., Iván R.-H., Erika D. L. C.-A., Carolina D. S., Keiko S., Alberto N.-Z. J.. 2021;Occurrence and infective potential of Colletotrichum gloeosporioides isolates associated to Citrus limon var Eureka. Biotechnol. Rep. 31:e00651.
Pramunadipta S., Widiastuti A., Wibowo A., Priyatmojo A.. 2022;rep-PCR analysis of Fusarium proliferatum causing sheath rot disease and its relationship to light, pH, temperature, and rice varieties. Arch. Phytopathol. Plant Prot. 55:973–990.
Prihastuti H., Cai L., Chen H., McKenzie E. H. C., Hyde K. D.. 2009;Characterization of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Divers. 39:89–109.
Ramos A. P., Talhinhas P., Sreenivasaprasad S., Oliveira H.. 2016;Characterization of Colletotrichum gloeosporioides, as the main causal agent of citrus anthracnose, and C. karstii as species preferentially associated with lemon twig dieback in Portugal. Phytoparasitica 44:549–561.
Saeed E. E., Sham A., AbuZarqa A., Al Shurafa K. A., Al Naqbi T. S., Iratni R., El-Tarabily K., AbuQamar S. F.. 2017;Detection and management of mango dieback disease in the United Arab Emirates. Int. J. Mol. Sci. 18:2086.
Savant N. V., Raut S. P.. 2000;Studies on symptomatology of die-back of mango stone grafts. Acta Hortic. 509:823–832.
Sharma G., Shenoy B. D.. 2016; Colletotrichum systematics: past, present and prospects. Mycosphere 7:1093–1102.
Siddiquee S.. 2017. Practical handbook of the biology and molecular diversity of Trichoderma species from tropical regions. Fungal biology Springer. Cham, Switzerland: p. 41–73.
Uysal A., Kurt S., Guarnaccia V.. 2022;Distribution and characterization of Colletotrichum species associated with Citrus anthracnose in eastern Mediterranean region of Turkey. Eur. J. Plant Pathol. 163:125–141.
Vitale A., Alfenas A. C., de Siqueira D. L., Magistà D., Perrone G., Polizzi G.. 2020;Cultivar resistance against Colletotrichum asianum in the world collection of mango germplasm in Southeastern Brazil. Plants 9:182.
Wang W., de Silva D. D., Moslemi A., Edwards J., Ades P. K., Crous P. W., Taylor P. W. J.. 2021; Colletotrichum species causing anthracnose of citrus in Australia. J. Fungi 7:47.
Widiastuti A., Suryanti Giovanni A. C., Santika I. A., Paramita N. R.. 2023;Fungal community associated with mixed infection of anthracnose and stem end rot diseases in Chokanan Mango. Biodiversitas 24:2163–2170.

Article information Continued

Fig. 1

Survey area of mango twig dieback in Java Island, Indonesia. The colored map was districts where the collection sample location was arranged.

Fig. 2

Dieback symptoms on mango tissue caused by Colletotrichum manifested as necrosis on branches, twigs, petioles, and leaves (A–C). The initial presentation included small, angular, brown-to-black specks that could expand into dried and lifeless areas. Arrows indicate the symptoms of dieback caused by Colletotrichum.

Fig. 3

DNA fingerprints of Colletotrichum spp. causing dieback on mango tree generated with primer sets BOX (A) and ERIC (B) for Colletotrichum spp. Lane 1, MLG-TJB; lane 2, HM-IJB; lane 3, GG-IJB; lane 4, MLG-IJB; lane 5, MLG-TJT; lane 6, HM-BDIY; lane 7, HM-SDIY; lane 8, HM-PAJT; lane 9, GAD-PAJT; lane 10, MLG-PAJT; lane 11, GO-PAJT; left M, 100 bp ladder Geneaid; right M, 1 kb ladder Geneaid.

Fig. 4

Dendrogram resulting from Unweighted Pair Group Method with Arithmetic Mean cluster analysis of DNA banding profiles generated with primer sets combined of BOX and ERIC for Colletotrichum spp. causing dieback on mango. The isolates in the red sign were selected for phylogenetic analysis.

Fig. 5

The phylogenetic tree of six representative isolates from this study was built using concatenated sequences of the internal transcribed spacer (ITS), partial actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) genes. Bootstrap support values from maximum likelihood above 50% were shown at each node. Monilochaetes infuscans CBS869.96 was used as the outgroup.

Fig. 6

Morphological characters of Colletotrichum asianum (isolate MLG-TJT) (A–E) and C. cairnsense (Isolate MLG-TJB) (F–J). (A, B, F, G) Colony of aerial and reverse on potato dextrose agar, 10 days growth from the single culture of conidia. (C, H) Conidiomata. (D, I) Conidia. (E, J) Appressoria and setae (setae only observed on C. asianum). Scale bars = 10 μm.

Fig. 7

Pathogenicity test of six isolates representative of Colletotrichum spp. causing dieback symptom on mango. (A) Control twig. (B, C) Dieback necrosis with a blackish lesion by cross-section on the tip of twig inoculated by C. asianum. (D, E) Dieback necrosis with a blackish lesion by cross-section lesion on the tip of twig inoculated by C. cairnsense. (F) Orange conidial mass captured by Lumix G Mirrorless (DSLM) Cameras DC-GH6 (Kadoma, Osaka, Japan). The arrows of A, B, and D indicate symptoms that appear at the point of inoculation. (G) Lesion area was measured in four perpendicular ways and means followed with the same letter are not significantly different on 95% confidence interval (P > 0.05) analyzed with Tukey honestly significant difference test.

Table 1

Colletotrichum spp. pure isolates used in this study

Isolate code Host (cultivar of mango) Origin

Province Subdistrict Latitude Longitude Altitude (m asl)
MLG-TJB Manalagi West Java Tasikmalaya −7.2251°S 108.1608°E 347
MLG-IJB Manalagi West Java Indramayu −6.3157°S 108.2001°E 11
GG-IJB Gedong Gincu West Java Indramayu −6.3085°S 108.1819°E 12
HM-IJB Arumanis West Java Indramayu −6.3157°S 108.2002°E 12
MLG-TJT Manalagi Central Java Temanggung −7.1638°S 110.0848°E 661
HM-SDIY Arumanis DI. Yogyakarta Sleman −7.4607°S 110.2253°E 140
HM-BDIY Arumanis DI. Yogyakarta Bantul −7.5015°S 110.2200°E 77
MLG-PAJT Manalagi East Java Pasuruan −7.4440°S 113.0134°E 48
GO-PAJT Golek East Java Pasuruan −7.4443°S 113.0136°E 49
GAD-PAJT Gadung East Java Pasuruan −7.4438°S 113.0134°E 45
HM-PAJT Arumanis East Java Pasuruan −7.4439°S 113.0134°E 45

m asl, meter above sea level.

Table 2

Colletotrichum spp. ex-type used in phylogenetic analysis

Species Isolate Host Country GenBank accession no.

ITS ACT GAPDH TUB2
C. asianum CMM4059 Mangifera indica Brazil KC329798 KC533750 KC517174 KC517283
C. asianum CMM4063 M. indica Brazil KC329771 KC533722 KC517175 KC517284
C. asianum CMM 4056 M. indica Brazil KC329789.1 KC533720.1 KC517165.1 KC517277.1
C. asianum CMM 4067 M. indica Brazil KC329803.1 KC533725.1 KC517179.1 KC517288.1
C. tropicale CMM 4073 M. indica Brazil KC329788.1 KC533730.1 KC517185.1 KC517262
C. tropicale CMM 4071 M. indica Brazil KC329785.1 KC533726.1 KC517181.1 KC517258.1
C. gloeosporioides UMC013 Citrus limon Australia MW081161 MW081167.1 MW081164.1 MW081170.1
C. gloeosporioides B1B3 Atractylodes ovata South Korea LC604483.1 LC604571.1 LC604545.1 LC604515.1
C. psidii CBS 145.29 Psidium sp. Italy JX010219.1 JX009515.1 JX009967.1 JX010443.1
C. cairnsense BRIP 63641 Capsicum annum Australia KU923671.1 KU923715.1 KU923703.1 KU923687.1
C. cairnsense AC11 Capsicum annum Australia KU318706.1 KU318709.1 KU318708.1 KU318707.1
C. acutatum IMI 223120 Anemone sp. Australia JQ948353.1 JQ949674.1 JQ948684.1 JQ950004.1
C. acutatum CBS 112979 Pinus radiata South Africa JQ948355.1 JQ949676.1 JQ948686.1 JQ950006.1
C. simmondsii CBS 122122 Carica papaya Australia JQ948276.1 JQ949597.1 JQ948606.1 JQ949927.1
C. simmondsii CBS 295.67 Fragaria sp. Australia JQ948278.1 JQ949599.1 JQ948608.1 JQ949929.1
C. Indonesiense CBS 127551 Eucalyptus sp. Indonesia JQ948288 JQ949609 JQ948618 JQ949939
C. cuscutae IMI 304802 Cuscuta sp. Dominica JQ948195 JQ949516 JQ948525 JQ949846
C. scovillei CBS 126529 Capsicum sp. Indonesia JQ948267.1 JQ949588.1 JQ948597.1 JQ949918.1
C. nymphaeae CBS:482.82 Protea sp. Australia JQ948213 JQ949534 JQ948543 JQ949864
C. nymphaeae CBS:126528 Capsicum annum Indonesia JQ948219 JQ949540 JQ948549 JQ949870
Monilochaetes infuscansa CBS869.96 Unknown USA NR_155365.1 JQ005843.1 JX546612.1 JQ005864.1

ITS, internal transcribed spacer; ACT, partial actin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TUB2, β-tubulin.

a

Outgroup.

Table 3

Characteristics of conidia and mycelial growth

Isolate code Conidiaa Mycelial growthc (mm/d)

Length × width (μm) L/Wb ratio
MLG-TJB (7–16.1) ± 1.94 × (2.8–5.1) ± 0.54 2.79 7.24 ± 0.09
HM-IJB (7.8–13.3) ± 1.31 × (2.9–5.2) ± 0.51 2.79 7.60 ± 0.11
MLG-TJT (10.9–16.4) ± 1.11 × (3.8–6.9) ± 0.58 2.74 7.30 ± 0.13
HM-BDIY (10.8–14.6) ± 0.97 × (2.9–5.5) ± 0.58 2.93 7.89 ± 0.01
MLG-PAJT (9.2–18.6) ± 1.79 × (3.8–6.6) ± 0.74 2.56 7.78 ± 0.02
HM-PAJT (8.6–15.3) ± 1.48 × (2.3–5.6) ± 0.64 3.23 7.69 ± 0.02
a

Conidia length and width were measured on 50 conidia for each isolate. The data are mean ± standard deviation.

b

Length per width.

c

Mycelial growth is measured in four perpendicular ways.