Introduction

India is known as the fruit and vegetable basket of the world (Yeledhalli et al. 2012). In the fiscal year 2012–13, India produced 81.29 million metric tons of fruits, 162.19 million metric tons of vegetables and 1.73 million metric tons of floricultural plants (Mistry et al. 2014). Indian fruits and vegetables are mainly exported to Middle East and South East Asian countries (DCGIS Annual Report 2014) and it is a major source of revenue for the economy (Kapila 2009). The pre- and post-harvest infections caused by Colletotrichum species result in severe losses in crop yield and quality, thus affect the export of fruits to other countries (Chadha 2009). It is essential that Colletotrichum species that cause anthracnose disease are accurately identified so as to develop effective disease-control strategies.

Colletotrichum gloeosporioides, in its traditional sense, was regarded as a major pre- and post-harvest pathogen causing anthracnose in economically important tropical crops. Recent taxonomic revisions reveal that C. gloeosporioides sensu lato is a species complex including 33 phylogenetic species and one subspecies (Lima et al. 2013; Liu et al. 2013; Manamgoda et al. 2013; Sharma et al. 2013; Udayanga et al. 2013; Vieira et al. 2014). It has been demonstrated that C. gloeosporioides sensu stricto is not a common pathogen of tropical fruits (Phoulivong et al. 2010; Sharma et al. 2013; Udayanga et al. 2013). Thus, it is important to revisit the old host-pathogen records and update it with molecular data as per recent nomenclatural revisions (Damm et al. 2012a, b; Weir et al. 2012). However, it is challenging to deal with the taxonomy of morphologically cryptic fungal species (Hibbett and Taylor 2013). This issue is very relevant and critical while dealing with identification of fungi that cause diseases in plants, animals and humans.

Colletotrichum siamense H. Prihastuti, L. Cai & K.D. Hyde, one of the challenging and controversial taxa, was described as a species associated with coffee berries by Prihastuti et al. (2009). In a major revision of the C. gloeosporioides species complex, Weir et al. (2012) later synonymised C. jasmini-sambac S. Wikee, K.D. Hyde, L. Cai and E. H. C. McKenzie (Wikee et al. 2011) and C. hymenocallidis Y.L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai (Yang et al. 2009) with C. siamense. Using the intergenic sequence of apn2 and Mat1-2 gene region (ApMat marker) and the translation elongation factor 1-α gene region (5′ tef1), Sharma et al. (2013), however, have demonstrated that C. jasmini-sambac, C. hymenocallidis, C. melanocaulon V.P. Doyle, P.V. Oudem. & S.A. Rehner (= C. dianesei N.B. Lima, M.P.S. Câmara & S. J. Michereff) (Vieira et al. 2014) and C. siamense are four distinct species within the C. siamense species complex. Some recent studies also support this hypothesis (Doyle et al. 2013; Udayanga et al. 2013; Vieira et al. 2014). Colletotrichum melanocaulon has recently been synonymised with C. dianesei and a new species C. endomangiferae W.A.S. Vieira, M.P.S. Câmara & S.J. Michereff has been described within the C. siamense species complex based on ApMat sequence data (Vieira et al. 2014). The species status of C. murrayae L.J. Peng & K.D. Hyde (Peng et al. 2012) within this complex is ambiguous, due to its illegitimate nomenclature (Liu et al. 2013).

Recent studies have demonstrated that ApMat marker is capable of efficient resolution of species within the C. gloeosporioides sensu lato (Rojas et al. 2010; Silva et al. 2012; Doyle et al. 2013) and C. siamense species complex (Sharma et al. 2013; Vieira et al. 2014). The occurrence of high level of fixed polymorphism among different species is believed to be responsible for the efficiency of the ApMat marker towards better phylogenetic species resolution (Silva et al. 2012). This study, therefore, aimed to unravel and describe novel Colletotrichum lineages/ taxa associated with anthracnose diseases of various host plants from India based on morphology, ApMat-marker phylogeny and pathogenicity data.

Materials and methods

Sample collection

Apparently healthy plant-tissue samples from selected ornamental and flowering plants (Bauhinia, Cassia, and Ficus) and guava (Psidium) fruits were collected from CSIR-Institute of Microbial Technology (CSIR-IMTECH) campus in Chandigarh. Symptomatic tea (Camellia) leaves were collected from tea gardens in Bir and Palampur regions of Kangra district in Himachal Pradesh. Banana (Musa) and orange (Citrus) fruits with lesions were procured from fruit-markets in Chandigarh. Banana fruits with lesions and neem (Azadirachta) leaves were purchased from a supermarket in Mysore, Karnataka. Infected coffee (Coffea) berries were collected and supplied by Mr. Deepak M. (CSIR-CFTRI, Mysore) from Virajpet Taluk in Karnataka. Fungal isolation from host-plant tissues was carried out as described by Cai et al. (2009).

Fungal isolates

Thirty-six isolates were recovered as endophytes or potential pathogens from the collected plant tissue samples as described above. One isolate was procured from Goa University Fungal Culture Collection (GUFCC), Goa. Two isolates were procured from National Fungal Culture Collection of India (NFCCI), Pune. Three isolates were accessed from the Microbial Type Culture Collection (MTCC), Chandigarh and 14 isolates from the Indian Type Culture Collection (ITCC), New Delhi. In addition, 29 isolates belonging to the C. siamense species complex associated with mango from our previous study (Sharma et al. 2013) were also included in this paper. Information on host and geographic location of sample collection of the isolates are detailed in Table 1. Fungal isolates were subcultured on potato dextrose agar (PDA, HiMedia, India) medium, grown at 20 °C for 7 days and preserved at −70 °C and liquid nitrogen in 10 % glycerol for future use.

Table 1 List of Colletotrichum isolates from this study with information on isolate designation, host, geographic location and GenBank accession numbers of the act, Apmat, cal, chs1, gapdh, ITS and tub2 gene sequences (# = Sequence not used in multigene phylogeny, N.A. information not available, N. S. not sequenced). All the isolates are of Indian origin
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DNA extraction, PCR amplification and sequencing of gene markers

Genomic DNA from fresh mycelia was isolated using the DNA isolation kit (catalogue number D6005, Zymo Research, USA) and stored at −20 °C. Fifty-six isolates from this study were subjected to polymerase chain reaction (PCR) amplification of the ApMat marker. Out of the 56 isolates, a subset of thirty-four Colletotrichum isolates was selected based on uniqueness of host and geographical location and subjected to PCR amplification of actin (act), calmodulin (cal), chitin synthase (chs1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), ITS and β-tubulin (tub2) gene regions. The reactions were carried out in an Eppendorf Mastercycler with the cycling parameters and primers as specified in previous papers (Damm et al. 2009–ITS, act, chs1, gapdh, tub2; Silva et al. 2012–ApMat and Weir et al. 2012–cal). The PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN, catalogue number 28106), quantified using Nanodrop Spectrophotometer ND-1000 (Thermo) and sequenced using respective forward and reverse primers with the ABI Big Dye v3.1 Terminator Ready Reaction Cycle Sequencing Kit (Applied Biosystems). Post sequencing reaction clean-up was performed to remove excess salt from samples, which were further denatured with HiDi-Formamide at 95 °C for 3 min and analysed using 3730 DNA Analyzer (Applied Biosystems) at the central DNA sequencing facility of CSIR-IMTECH, Chandigarh. The sequences generated in this study are deposited in NCBI-GenBank with accession numbers as listed in Table 1.

ApMat marker-based phylogenetic analysis

Fifty-six Colletotrichum isolates belonging to the C. gloeosporioides species complex were selected for this analysis. Twenty-nine sequences belonging to the C. siamense species complex and associated with mango tissues were retrieved from Sharma et al. (2013). Information on GenBank accession numbers is detailed in Tables 1 and 2. A maximum parsimony (MP) analysis of the ApMat dataset was performed using PAUP version 4.0b10 (Swofford 2003). The ambiguously aligned regions were not included in the analysis. The gaps in the alignment were treated as missing data. All the characters in the analysis were unordered and had equal weight. Trees were inferred using the heuristic search option with 20 random sequence additions and tree bisection and reconstruction (TBR) as the branch swapping algorithm. Maxtrees were set to 10.000; the branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics such as Tree Length (TL), Consistency Index (CI), Retention Index (RI), Related Consistency Index (RCI) and Homoplasy Index (HI) were calculated for the generated trees. The clade stability was assessed by 100 bootstrap replicates (Felsenstein 1985) and addition of 10 random sequences. Kishino-Hasewaga tests (Kishino and Hasewaga 1989) were performed to determine whether trees were significantly different. Trees were viewed in TreeView version 1.6.6 (Page 1996) and edited in MEGA version 5.2 (Tamura et al. 2011) and Microsoft PowerPoint version 2007 (Microsoft Corp. USA). The alignment file and tree are deposited in TreeBase (www.treebase.org; Study ID: 14671).

Table 2 List of GenBank accession numbers of the ApMat sequences from Sharma et al. (2013) used in this study with information on taxa and isolate designation. All the isolates are from mango samples collected from India
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5-gene based phylogenetic analysis of the C. siamense species complex

A multigene dataset including: newly generated act, cal, gapdh, ITS and tub2 gene sequences of 23 isolates of the C. siamense species complex and the homologous sequences retrieved from GenBank of seven reference taxa was prepared using SequenceMatrix version 1.7.8 (Vaidya et al. 2011). The sequences from the ex-type isolates of C. dianesei, C. endomangiferae, C. hymenocallidis, C. jasmini-sambac, C. murrayae and C. siamense were included in the analysis. All the details related to gene sequences are presented in Tables 1 and 3. The chs1 gene region was not included in the multigene dataset as it was missing for the following taxa: C. dianesei and C. murrayae. The MP analysis of the 5-gene dataset was performed using PAUP as described in ApMat marker based phylogenetic analysis. The alignment files and trees are deposited in TreeBase (www.treebase.org; Study ID: 14671).

Table 3 List of GenBank accession numbers for the ITS, gapdh, cal, act, chs1, tub2 and ApMat gene sequences of the ex-type isolates belonging to the Colletotrichum gloeosporioides species complex with information on taxa, host and geographic location
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Morphological characterisation

For selected isolates (Table 4), morphological characterisation was carried out based on the 7-day old cultures grown at 20 °C on PDA. Micro-morphological characters such as colour, shape and size of conidia and conidiogenous cells were observed and photographed using a trinocular differential interference contrast (DIC) microscope (Olympus U-CMAD3) equipped with an Olympus microscope camera. For each isolate, length and width of at least 30 randomly chosen conidia were measured using the CellB image analysis software (Olympus). The colony diameter was measured after 7 days to determine the growth rate (mm/ day). Selected morphological features of the isolates are listed in Table 4.

Table 4 Comparison of morphological characters of selected Colletotrichum isolates with the ex-type isolates (*)
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Pathogenicity testing

Pathogenicity testing was performed as outlined in Sharma et al. (2013). Following representative isolates were selected for pathogenicity tests: Camellia–G. cingulata f. sp. camelliae (TB01 = MTCC 11728); Citrus–C. communis sp. nov. (GO01 = MTCC 11696), Psidium–C. communis sp. nov. (MTCC 4626); Mangifera–C. communis sp. nov. (NK24 = MTCC 11599); Mangifera–C. endomangiferae (GM529 = MTCC 11592, GM473 = MTCC 11589); Musa–C. aotearoa (GBM2 = MTCC 11769), C. musae (GBM3 = MTCC 11768). Fresh, unripe fruits and leaves were surface-sterilized using 1 % sodium hypochlorite solution and wounded using a sterile needle. Four fruits/ leaves were inoculated with 6 μl of conidial suspension (1 × 106 spore/ ml) for each isolate and one was inoculated with sterile water and used as control. The fruits/ leaves were kept in a moist chamber. The appearance of disease symptoms was observed from 4 to 7 days of incubation at 20 °C (Figs. 3 and 4). Severity of disease was calculated by measuring the lesion size and scored on a 0–9 point scale based on the percentage of the infected area (Montri et al. 2009). Percent disease incidence (PDI) and percent disease severity (PDS) were calculated using the formula given below (Awa et al. 2012) and the resulting values for PDI and PDS are shown in Table 5.

Table 5 Disease score (DS) on a 0–9 scale for each fruit/ leaf and value of percent disease incidence (PDI) and percent disease severity (PDS) (* = ex-type isolate)
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$$ \mathbf{P}\mathbf{D}\mathbf{I}\left(\%\right)=\mathbf{x}/\mathbf{N}\times \mathbf{100} $$
$$ \mathbf{P}\mathbf{D}\mathbf{S}\left(\%\right)=\left[\varSigma\ \left(\mathbf{a}+\mathbf{b}\right)/\mathbf{N}\times \mathbf{Z}\right]\times \mathbf{100} $$

Where; Σ (a+b) = Sum of score scales of all inoculated host tissue samples

N:

Total number of inoculated host tissue samples

Z:

Highest score scale

X:

Number of infected host tissue samples

Results

ApMat marker-based phylogenetic analysis

The ApMat dataset included 114 sequences and a total of 955 characters including gaps. Fifty-one characters from the ambiguously-aligned regions were excluded from the analysis. Out of the remaining 904 characters, 409 characters were constant, 331 characters were parsimony-informative and 164 characters were parsimony-uninformative. The MP analysis resulted in 33 trees and based on the KH test, these trees were not significantly different (details not shown). One of the 33 trees (TL = 815, CI = 0.758, RI = 0.939, RC = 0.712, HI = 0.242) generated in the MP analysis is shown in Fig. 1. The tree is rooted with C. xanthorrhoeae ICMP 17903. The bootstrap support values more than 50 % for the observed branching pattern are shown next to the branches. In the MP tree shown in Fig. 1, bootstrap support for majority of the clades is higher than 70 %. The ApMat analysis resolved most of the isolates to their species level. Seven known species (C. aotearoa, C. dianesei, C. endomangiferae, C. musae, C. siamense, C. theobromicola and G. cingulata f. sp. camelliae) and six novel lineages (designated as C. communis sp. nov. and Colletotrichum sp. indet. 1–5) were recovered in this analysis.

Fig. 1
figure 1

One of the 33 most parsimonious trees showing phylogenetic affinities of 85 Colletotrichum isolates from India (highlighted in blue), obtained from heuristic search of the ApMat dataset. Colletotrichum xanthorrhoeae ICMP 17903 is the designated outgroup. Bootstrap support values of more than 50 % are shown at the nodes. Ex-type isolates are marked with *

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Colletotrichum isolates ITCC 6161 and ITCC 6164 clustered with the ex-type isolate of C. theobromicola (ICMP 18649). Colletotrichum isolate GBM2 clustered with the ex-type isolate of C. aotearoa (ICMP 18537). Colletotrichum isolates GB07, GB15 and GBM3 clustered with the ex-type isolate of C. musae (ICMP 19119). Colletotrichum isolates MTCC 11728, MTCC 11729, MTCC 11730 and MTCC 11731 associated with tea leaves clustered as a distinct clade. In the 5-gene analysis these four isolates clustered with the representative strain of G. cingulata f. sp. camelliae (ICMP 18542) (data not shown). However, in the ApMat analysis the representative sequence could not be included due to unavailability of ex-type isolate. Epitypification of Glomerella cingulata f. sp. camelliae is pending and expected to be completed soon (Lei Cai, personal communication).

In the C. siamense species complex, Colletotrichum isolate GN01 clustered with the ex-type isolate of C. siamense sensu stricto (ICMP 18578). The isolates GS02, GS07, GS20, IMTF976, IMTF997, ITCC 4981, MTCC 9663 from this study and the isolates GM057, GM063, GM172, GM291, GM388, GM409, GM514 from Sharma et al. (2013) clustered with the ex-type isolate of C. dianesei (MFLU 1300058). The isolates GUFCC 15502, NFCCI 1737 from this study and GM473, GM529, MTCC 9660 from Sharma et al. (2013) clustered with the ex-type isolate of recently described C. endomangiferae (CMM3814).

A strongly supported clade including isolates GO01–06, GS01, GS03, GS06, GS14, GS17–19, GS21, GS22, GS28, GS29, IMTF 736–738, ITCC 5123, ITCC 6152, ITCC 6153, ITCC 6155, ITCC 6159, ITCC 6336, MTCC 4626 and NFCCI 1925 from this study and isolates GM010, GM018, GM043A, GM043B, GM147, GM150, GM192, GM301, GM314, GM397, ITCC 6158, NK22–25 and NK28–29 from Sharma et al. (2013) was recovered as a novel lineage. This novel lineage is described in this paper as C. communis sp. nov. In addition, five novel lineages designated as Colletotrichum sp. indet 1–5 (Fig. 1) were recovered in this analysis. These novel lineages could not be described as new taxa due to inability of the isolates to sporulate or availability of only one representative isolate.

5-gene based phylogenetic analysis of the C. siamense species complex

The multigene dataset included 2253 characters including gaps. The gene boundaries in the dataset included: ITS: 1–576, act: 577–834, tub2: 835–1258, cal: 1259–1998 and gapdh: 1999–2253. The analysis involved 30 isolates. Twenty-one characters from the ambiguously-aligned regions were excluded from the analysis. Out of the remaining 2232 characters, 2066 characters were constant, 36 characters were parsimony-informative and 130 characters were parsimony-uninformative. The MP analysis resulted in 90 trees and based on the KH test, these trees were not significantly different (details not shown). One of the 90 trees (TL = 206, CI = 0.830, RI = 0.696, RC = 0.577, HI = 0.170) generated in the MP analysis is shown in Fig. 2. The tree is rooted with C. gloeosporioides ICMP 17821. The bootstrap support values more than 50 % for the observed branching pattern are shown next to the branches. The MP tree shown in Fig. 2 is poorly supported.

Fig. 2
figure 2

One of the 90 most parsimonious trees showing phylogenetic affinities of 23 isolates of the C. siamense species complex from India (highlighted in blue), obtained from heuristic search of the 5-gene dataset (act, cal, gapdh, ITS and tub2). Colletotrichum gloeosporioides ICMP 17821 is the designated outgroup. Bootstrap support values of more than 50 %, are shown at the nodes. Ex-type isolates are marked with *

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Morphological comparison

The comparison of morphological characters (colony morphology on PDA, conidial measurements and shape) and growth rate is presented in Table 4. There were no apparent differences in the morphotaxonomic characters.

Taxonomy

Colletotrichum communis Sharma G., Pinnaka, A. K. & Shenoy, B. D., sp. nov. (Fig. 4)

MycoBank No.: MB808066

Etymology: named after the prevalent (common) distribution of this species over different tropical host plants

Description: Colonies on PDA attaining 74–91 mm diam. after 7 days at 20 °C, growth rate 10.6–13.0 mm per day (n = 10), whitish to greyish black, reverse light orange to dark grey at the centre, aerial mycelium, dense, with orange conidial mass. Sexual stage not observed on PDA plate. Asexual stage widely observed. Conidiomata acervular. Conidiophores hyaline, septate, branched at base, smooth. Conidiogenous cells 14–18 μm in length (n = 20) and 2–3 μm in width, enteroblastic, phialidic, hyaline, cylindrical or clavate shaped, the base is slightly wider than the apex, hyaline, arranged in clusters, unbranched. Conidia 11.1–18.2 μm in length and 3.7–7.3 μm in width (n = 100), hyaline, smooth, cylindrical with rounded ends. Appressoria rarely formed on PDA, brown to dark brown, shape variable, 5–10 μm in length and 4–7 μm in width (n = 10). Setae formed, dark brown to black in color, smooth, tapered towards apices, 70–90 μm in length and 2–5 μm in width (n = 10).

Geographic distribution and host range: Bauhinia variegata (orchid tree), Cassia fistula (golden shower tree), Cassia sp., Citrus sp. (orange), Ficus elastica (rubber plant), Mangifera indica (mango), Psidium guajava (guava) and Saraca indica (ashoka tree) in different locations of India as mentioned in Table 1.

Materials examined: INDIA, Udupi district (13° 20′ N 74° 44′ E) in Karnataka state, on fruit lesion symptoms of Mangifera indica (mango) of Neelam variety, June 2011, Gunjan Sharma & Belle Damodara Shenoy (NK24* = MTCC 11599*, ex-type culture).

Additional specimens examined: INDIA, Chandigarh (U.T.) (30° 45′ N 76° 47′ E), on fruit spots of Citrus, December 2012, Gunjan Sharma (GO01 = MTCC 11696); INDIA, Chandigarh (U.T.) (30° 45′ N 76° 47′ E), from fruit lesions of Guava, Sandeep Jain (MTCC 4626).

Notes: Colletotrichum communis sp. nov. is morphologically similar to C. siamense but the former has a higher growth rate and slightly longer conidia. Colletotrichum communis sp. nov. is described as a pathogenic species associated with a broad host range in India based on the ApMat sequences.

Pathogenicity testing

The fruits/ leaves inoculated with conidial suspension of selected Colletotrichum isolates developed typical dark brown lesions of anthracnose disease around the wound (Figs. 3 and 4). The pathogens were re-isolated from the infected host tissues on to PDA medium to confirm the Koch’s postulates. However, the control did not develop any symptoms after 7 days of inoculation. The results of pathogenicity testing are provided in Table 5. Based on the percent disease severity (PDS) calculations, C. communis sp. nov. proved to be highly pathogenic to the tropical fruits tested in this study. It produced anthracnose lesions on Citrus (Orange), Mangifera (Mango) and Psidium (Guava) fruits with 72.2, 77.7 and 83.3 % severity. Colletotrichum endomangiferae produced anthracnose lesions on mango fruits with 61.1–72.2 % severity. Colletotrichum aotearoa isolate was slightly pathogenic to Musa (banana) with 11.1 % disease severity while banana anthracnose pathogen C. musae was moderately pathogenic with 55.5 % disease severity. Tea anthracnose pathogen G. cingulata f. sp. camelliae isolate from this study was found to be moderately pathogenic with 38.8 % disease severity, in the capacity to cause anthracnose lesion on Camellia (tea) leaves.

Fig. 3
figure 3figure 3

Morphology (after 7 days on PDA) and results of pathogenicity testing of selected isolates (a–f) on selected hosts i. Colony morphology (front), ii. Colony morphology (reverse), iii. Conidia (scale bar =10 μm), iv. Control fruit/ leaf, v. Fruit/ leaf seven days after inoculation

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Fig. 4
figure 4

Morphological features of C. communis sp. nov. (NK24* = MTCC 11599*) and results of pathogenicity testing i-xii Morphological features, i Colony morphology on PDA (front), ii Colony morphology on PDA (reverse), iii Conidiogenous cells, iv-v Setae, vi Conidia, vii-xii Appresoria (Scale bar of iii-vi =20 μm, Scale bar of vi applies to vii-xii), xiii-xiv Results of pathogenicity testing, xiii Control mango fruit, xiv symptoms 7 days after infection

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Discussion

Based on ApMat marker analysis, this study has established that the C. siamense species complex includes six previously known species (C. dianesei, C. endomangiferae, C. hymenocallidis, C. jasmini-sambac, C. murrayae and C. siamense) and one novel species C. communis sp. nov. (Table 6). We accept C. hymenocallidis Y.L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai, C. jasmini-sambac S. Wikee, K.D. Hyde, L. Cai and E. H. C. McKenzie and C. siamense H. Prihastuti, L. Cai & K.D. Hyde as distinct species within the C. siamense species complex. Five novel lineages (potential new species), designated in this paper as Colletotrichum sp. indet 1–5, recovered within the C. siamense species complex (Table 6) are subject to further investigation with more isolates from diverse hosts.

Table 6 List of species in the Colletotrichum siamense species complex with information on host and geographic distribution
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The members of C. siamense species complex have been reported from several plants hosts (Yang et al. 2009; Wikee et al. 2011; Yang et al. 2011; Weir et al. 2012; Li et al. 2012; Cheng et al. 2013; Lima et al. 2013; Doyle et al. 2013; Sharma et al. 2013; Udayanga et al. 2013; James et al. 2014; Vieira et al. 2014). This study further reports the association of the members of C. siamense species complex with ashoka tree (Saraca), coconut (Cocos), coffee (Coffea), dumb cane (Dieffenbachia), golden shower tree (Cassia), guava (Psidium), mango (Mangifera), neem (Azadirachta), orange (Citrus), orchid tree (Bauhinia), papaya (Carica), pomegranate (Punica) and rubber plant (Ficus) in India. Additionally, this study reports C. aotearoa and C. musae from banana (Musa), C. theobromicola from pomegranate and G. cingulata f. sp. camelliae from tea (Camellia).

Though Colletotrichum aotearoa has been reported from New Zealand on a wide variety of native host plants (Weir et al. 2012), this is the first report on its association with banana fruits (Musa sp.). Colletotrichum musae is widely known as the pathogen of banana anthracnose (Abd-Elsalam et al. 2010; Su et al. 2011), but lesser known species such as C. karstii (Damm et al. 2012a) and C. paxtonii (Damm et al. 2012b) have also been associated with banana fruit. The pathogenic potential of C. karstii and C. paxtonii is poorly understood. In this study, we performed pathogenicity testing of C. aotearoa isolate (GBM2 = MTCC 11769) on banana fruits and the isolate exhibited a low pathogenic potential with 11.1 % disease severity, as compared to the C. musae isolate GBM3 with 55.5 % disease severity.

This study reports G. cingulata f. sp. camelliae from symptomatic tea (Camellia sinensis) leaves from tea gardens of Kangra district in Himachal Pradesh, India. As discussed in Weir et al. (2012), it was observed in our analysis that G. cingulata f. sp. camelliae isolates form a well-supported lineage within the Kahawae clade, both in the 5-gene (data not shown) and the ApMat marker based phylogenetic analyses (Fig. 1). The name Glomerella cingulata f. sp. camelliae was used by Dickens and Cook (1989) for the C. gloeosporioides sensu lato isolates associated with Camellia twig blight. This species has been reported from different Camellia hosts such as Ca. japonica, Ca. oleifera, Ca. reticulata, Ca. saluenensis, Ca. sasanqua, Ca. sinensis and Ca. × williamsii from Australia, China, France, Italy, Japan, Kenya, Korea, Malaysia, Taiwan, Tanzania, UK, USA and Zimbabwe (Weir et al. 2012; Farr and Rossman 2014). However, the epitypification of this species is pending (Lei Cai, personal communication) and thus the identification of the tea isolates from this study is not definite in the absence of a valid type material.

This study has focussed on identification and description of cryptic species within the C. siamense species complex from India. Due to observed low level of genetic divergence among species complexes, we recommend the use of “powerful gene markers” such as ApMat marker. This will not only be time-saving, but also cost-effective in comparison with sequencing and analysing 5–8 genes. We promote the ApMat marker as an efficient marker for finer phylogenetic resolution within the C. siamense species complex and C. gloeosporioides sensu lato. There is a need to strengthen the ApMat dataset by sequencing missing type strains, so that a comprehensive phylogenetic analysis could be done by the researchers in future. A consensus among the researchers on gene markers to be used while describing a new Colletotrichum species is desirable.