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Research Article
Morphological and molecular phylogenetic analyses reveal three species of Colletotrichum in Shandong province, China
expand article infoTaichang Mu, Zhaoxue Zhang, Rongyu Liu, Shubin Liu, Zhuang Li, Xiuguo Zhang, Jiwen Xia
‡ Shandong Agricultural University, Tai'an, China
Open Access

Abstract

Colletotrichum has numerous host range and distribution. Its species are important plant pathogens, endophytes and saprobes. Colletotrichum can cause regular or irregular depressions and necrotic lesions in the epidermal tissues of plants. During this research Colletotrichum specimens were collected from Mengyin County, Shandong Province, China. A multi-locus phylogenetic analysis of ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS sequence data combined with morphology, revealed a new species and two known species, viz. C. mengyinense sp. nov., C. gloeosporioides and C. pandanicola, belonging to the C. gloeosporioides species complex. The new species is described and illustrated in this paper and compared with taxa in the C. gloeosporioides species complex.

Keywords

Colletotrichum, Glomerellaceae, multi-gene phylogeny, new species, taxonomy

Introduction

Colletotrichum species (Glomerellaceae, Glomerellales) is one of the ten economically most important fungal plant pathogens worldwide (Dean et al. 2012). It was first observed by Tode (1790), who divided it into Vermicularia. Corda (1831) established Colletotrichum based on the characteristic of the conidiomata with setae in Vermicularia. Colletotrichum is based on the type species Colletotrichum lineola which was associated with a member of the Apiaceae (Jayawardena et al. 2017). The sexual morph belongs to Glomerella. The asexual morph is characterized by acervuli born in the skin of the host, often producing brown sharp setae, colorless or brown conidiophores with separate, conidia colorless, pseudomonas, cylindrical or crescent-shaped (Damm et al. 2009).

Currently, more than 900 epithets of Colletotrichum are listed in Index Fungorum (http://www.indexfungorum.org/; accessed 22 November 2021). Colletotrichum has been studied for more than 200 years and the classification of Colletotrichum has undergone major changes (Jayawardena et al. 2016). In order to clarify its complex nature, the species are classified into 14 species complexes (Bhunjun et al. 2021). Specifically, C. gloeosporioides has been considered as a complex species for a long time.

The name C. gloeosporioides was first proposed by Penzig based on Vermicularia gloeosporioides which was collected from Citrus in Italy (Weir et al. 2012). Early in the study of C. gloeosporioides species complex, taxonomic concepts used were based on apparent features such as morphological characters, host species, size and shape of conidia and appressoria, presence or absence of setae, aspect, color and growth rate in culture, whether or not the teleomorph develops, etc (Weir et al. 2012). Nonetheless, Sutton commented that “no progress in the systematics and identification of isolates belonging to this complex is likely to be made based on morphology alone”. Fortunately, with the development of molecular systematics, gene method is applied to taxonomy of Colletotrichum complexes. Multi-gene phylogeny analysis is of great significance to the study of the classification of C. gloeosporioides species complex and related concepts of species (Cannon et al. 2012; Damm et al. 2012; Weir et al. 2012).

The aim of this study was to explore the diversity of Colletotrichum species from symptomatic leaves and diseased fruit of plants in Shandong Province, China. We present a new species and two known species, C. mengyinense sp. nov., C. gloeosporioides and C. pandanicola based on phylogenetic data and morphology.

Materials and methods

Isolation and morphological studies

The samples were collected from Mengyin County, Shandong Province, China. The strains of Colletotrichum were isolated from symptomatic leaves of Rosa chinensis and diseased fruit of Juglans regia using single spore and tissue isolation methods (Chomnunti et al. 2014). The spore suspension was obtained and spread onto PDA plate and incubated for one day under the biochemical incubator. After germination, the spores were transferred to a new PDA plate to obtain pure culture. Additionally, the surface sterilized plant tissue isolation was used to obtain sterile isolates from the host plant. About 25 mm2 tissue fragments were taken from the margin of tissue lesions and surface sterilized by consecutively immersing in 75% ethanol solution for 60 s, 5% sodium hypochlorite solution for 30 s, and then rinsed in sterile distilled water for 60 s (Gao et al. 2013; Liu et al. 2015). The surface sterilized plant tissue was dried with sterilized paper and moved on the PDA plate (Cai et al. 2009). All the PDA plates were incubated at biochemical incubator at 25 °C for 3–4 days, then hyphae were picked out of the periphery of the colonies and inoculated on to new PDA plates.

Following 5–14 days of incubation, morphological characters were recorded (Cai et al. 2009). Photographs of the colonies were taken at 7 days and 14 days using a digital camera (Canon G7X). Micromorphological characters of colonies were observed using stereomicroscope (Olympus SZX10) and microscope (Olympus BX53), both fitted with high definition color digital cameras to photo document conidia and so on of fungal structures. All Colletotrichum strains were stored in 10% sterilized glycerin and sterile water at 4 °C for deep studies in the future. Every specimen was deposited in the Herbarium of the Department of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from Colletotrichum fungal mycelia grown on PDA after 5–7 days, using a modified cetyltrimethylammonium bromide (CTAB) buffer, and then it was incubated at 65 °C for 30 min with occasional gentle inverting (Guo et al. 2000). Gene sequences were obtained from seven genes loci including the internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), partial glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), partial chitin synthase 1 gene (CHS-1), partial actin gene (ACT), partial beta-tubulin gene (TUB2), partial calmodulin gene (CAL) and partial glutamine synthetase gene (GS) were amplified and sequenced using primers pairs (Table 1).

Table 1.

Gene regions and respective primer pairs used in the study.

Locus Gene Primer Direction Sequence (5'-3')
The internal transcribed spacer regions with intervening 5.8S nrRNA gene ITS ITS5 Forward GGA AGT AAA AGT CGT AAC AAG G
ITS4 Reverse TCC TCC GCT TAT TGA TAT GC
Partial glyceraldehyde-3-phosphate dehydrogenase gene GAPDH GDF1 Forward GCC GTC AAC GAC CCC TTC ATT GA
GDR1 Reverse GGG TGG AGT CGT ACT TGA GCA TGT
Partial chitin synthase 1 gene CHS-1 CHS-79F Forward TGG GGC AAG GAT GCT TGG AAG AAG
CHS-354R Reverse TGG AAG AAC CAT CTG TGA GAG TTG
Partial actin gene ACT ACT-512F Forward ATG TGC AAG GCC GGT TTC GC
ACT-783R Reverse TAC GAG TCC TTC TGG CCC AT
Partial beta-tubulin gene TUB2 Bt-2a Forward GGT AAC CAA ATC GGT GCT GCT TTC
Bt-2b Reverse ACC CTC AGT GTA GTG ACC CTT GGC
Partial calmodulin gene CAL CL1 Forward GAR TWC AAG GAG GCC TTC TC
CL2A Reverse TTT TTG CAT CAT GAG TTG GAC
CL1C Forward GAA TTC AAG GAG GCC TTC TC
CL2C Reverse CTT CTG CAT CAT GAG CTG GAC
Partial glutamine synthetase gene GS GSLF3 Forward GAT ACG CCT CTT CCA GCG TT
GSLR1 Reverse AGR CGC ACA TTG TCA GTA TCG

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume which contained 12.5 μL 2× Taq Plus Master Mix II (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Tsingke, Qingdao, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL. PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The annealing temperature for each gene was 52 °C for ITS and GS, 59 °C for CAL, 60 °C for GAPDH, 58 °C for ACT and CHS-1, 55 °C for TUB2. The PCR products were visualized on 1% agarose electrophoresis gel. Sequencing was conducted by the Tsingke Company Limited (Qingdao, China) bi-directionally. Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 2).

Table 2.

Species and GenBank accession numbers of DNA sequences used in this study with new sequences in bold.

Species Strain/Isolate Host/Substrate GenBank accession number
ITS GAPDH CHS-1 ACT TUB2 CAL GS
Colletotrichum aenigma ICMP 18608* Persea americana JX010244 JX010044 JX009774 JX009443 JX010389 JX009683 JX010078
C. aeschynomenes ICMP 17673*=ATCC 201874 Aeschynomene virginica JX010176 JX009930 JX009799 JX009483 JX010392 JX009721 JX010081
C. alatae CBS 304.67*=ICMP 17919 Dioscorea alata JX010190 JX009990 JX009837 JX009471 JX010383 JX009738 JX010065
C. alienum ICMP 12071* Malus domestica JX010251 JX010028 JX009882 JX009572 JX010411 JX009654 JX010101
C. aotearoa ICMP 18735 Hedychium gardnerianum JX010221 JX010023 JX009880 JX009500 JX010424 JX009620 JX010115
C. arecicola hb8 Areca catechu MW561344 MW557464 - - MW557482 - -
C. artocarpicola MFLUCC18-1167* Artocarpus heterophyllus MN415991 MN435568 MN435569 MN435570 MN435567 - -
C. asianum ICMP 18580*=CBS 130418 Coffea arabica FJ972612 JX010053 JX009867 JX009584 JX010406 FJ917506 JX010096
C. australianum BRIP 63695 Capsicum annuum KU923677 MN442115 MW092000 MN442105 KU923693 - KU923737
C. boninense (outgroup) CBS 123755* Crinum asiaticum var. sinicum JQ005153 JQ005240 JQ005327 JQ005501 JQ005588 - -
C. camelliae ICMP 10643 Camellia × williamsi JX010224 JX009908 JX009891 JX009540 JX010436 JX009630 JX010119
C. changpingense MFLUCC 15-0022* Fragaria × ananassa KP683152 KP852469 KP852449 KP683093 KP852490 - -
C. chiangmaiense MFLUCC 18-0945 Magnolia garrettii MW346499 MW548592 MW623653 MW655578 - - -
C. chrysophilum CMM4268* Musa sp. KX094252 KX094183 KX094083 KX093982 KX094285 KX094063 KX094204
C. ciggaro ICMP 19122 Vaccinium sp. JX010228 JX009950 JX009902 JX009536 JX010433 JX009744 JX010134
C. clidemiae ICMP 18658* Clidemia hirta JX010265 JX009989 JX009877 JX009537 JX010438 JX009645 JX010129
C. cobbittiense BRIP66219 Cordyline stricta × Cordyline australis MH087016 MH094133 MH094135 MH094134 MH094137 - -
C. conoides CAUG17* Capsicum annuum KP890168 KP890162 KP890156 KP890144 KP890174 - -
C. cordylinicola MFLUCC090551*=ICMP 18579 Cordyline fruticosa JX010226 JX009975 JX009864 HM470235 JX010440 HM470238 JX010122
C. dracaenigenum MFLUCC 19-0430* Dracaena fragrans MN921250 MT215577 MT215575 MT313686 - - -
C. endophytica CAUG28 Capsicum annuum KP145441 KP145413 KP145385 KP145329 KP145469 - -
C. fici-septicae MFLU 19-27708* Ficus septica MW114367 MW183774 MW177701 MW151585 - - -
C. fructicola MFLU 090228* Coffea arabica FJ972603 FJ972578 - FJ907426 FJ907441 FJ917508 FJ972593
C. fructivorum CBS 133125* Vaccinium macrocarpon JX145145 - - - JX145196 - -
C. gloeosporioides IMI356878*=ICMP 17821 Citrus sinensis JX010152 JX010056 JX009818 JX009531 JX010445 JX009731 JX010085
ICMP 19121 Citrus limon JX010148 JX010054 JX009903 JX009558 - JX009745 -
SAUCC200952 Juglans regia MW786743 MW876474 MW883689 MW883698 MW888973 MW922541 MW888964
SAUCC200954 Juglans regia MW786744 MW876475 MW883690 MW883699 MW888974 MW922542 MW888965
SAUCC201001 Juglans regia MW786745 MW876477 MW883692 MW883701 MW888976 MW922544 MW888967
C. grevilleae CBS 132879* Grevillea sp. KC297078 KC297010 KC296987 KC296941 KC297102 KC296963 -
C. grossum CAUG7* Capsicum sp. KP890165 KP890159 KP890153 KP890141 KP890171 KP890147 -
C. hebeiense MFLUCC130-726* Vitis vinifera KF156863 KF377495 KF289008 KF377532 KF288975 - -
C. hedericola MFLU 15-0689 Hedera helix MN631384 - MN635794 MN635795 - - -
C. helleniense CBS 142418* Poncirus trifoliata KY856446 KY856270 KY856186 KY856019 KY856528 - -
C. henanense LF238* Camellia sinensis KJ955109 KJ954810 - KM023257 KJ955257 KJ954662 KJ954960
C. horii ICMP 10492 Diospyros kaki GQ329690 GQ329681 JX009752 JX009438 JX010450 JX009604 JX010137
C. hystricis CPC 28153* Citrus hystrix KY856450 KY856274 KY856190 KY856023 KY856532 - -
C. jiangxiense LF687* Camellia sinensis KJ955201 KJ954902 - KJ954471 KJ955348 KJ954752 KJ955051
C. kahawae IMI 319418*=ICMP 17816 Coffea arabica JX010231 JX010012 JX009813 JX009452 JX010444 - JX010130
C. ledongense CGMCC3.18888* Quercus palustris MG242008 MG242016 MG242018 MG242014 MG242010 - -
C. makassarense CBS 143664a*=CPC 28612 Capsicum annuum MH728812 MH728820 MH805850 MH781480 MH846563 - -
C. mengyinense SAUCC200702* Rosa chinensis MW786742 MW846240 MW883686 MW883695 MW888970 MW922538 MW888961
SAUCC200912 Juglans regia MW786689 MW876472 MW883687 MW883696 MW888971 MW922539 MW888962
SAUCC200913 Juglans regia MW786690 MW876473 MW883688 MW883697 MW888972 MW922540 MW888963
SAUCC200983 Juglans regia MW786642 MW876476 MW883691 MW883700 MW888975 MW922543 MW888966
C. musae CBS 116870*=ICMP 19119 Musa sp. JX010146 JX010050 JX009896 JX009433 HQ596280 JX009742 JX010103
C. nupharicola CBS 470.96*=ICMP 18187 Nuphar lutea subsp. polysepala JX010187 JX009972 JX009835 JX009437 JX010398 JX009663 JX010088
C. pandanicola MFLU 18-0003* Pandanus sp. MG646967 MG646934 MG646931 MG646938 MG646926 - -
SAUCC200204 Juglans regia MW786641 MW846239 MW883685 MW883694 MW888969 MW922537 MW888960
SAUCC201152 Juglans regia MW786746 MW876478 MW883693 MW883702 MW888977 MW922545 MW888968
C. perseae GA100* Persea americana KX620308 KX620242 - KX620145 KX620341 KX620206 KX620275
C. proteae CBS 132882* Protea sp. KC297079 KC297009 KC296986 KC296940 KC297101 KC296960 -
C. pseudotheobromicola MFLUCC 18-1602 Prunus avium MH817395 MH853675 MH853678 MH853681 MH853684 - -
C. psidii ICMP 19120 Psidium sp. JX010219 JX009967 JX009901 JX009515 JX010443 JX009743 JX010133
C. queenslandicum ICMP 1778* Carica papaya JX010276 JX009934 JX009899 JX009447 JX010414 JX009691 JX010104
C. rhexiae CBS 133134* Rhexia virginica JX145128 - - - JX145179 - -
C. salsolae ICMP 19051* Salsola tragus JX010242 JX009916 JX009863 JX009562 JX010403 - -
C. siamense ICMP 18578* Coffea arabica JX010171 JX009924 JX009865 FJ907423 JX010404 FJ917505 JX010094
ICMP 19118 Jasminum sambac HM131511 HM131497 JX009895 HM131507 JX010415 - JX010105
C. syzygicola MFLUCC10-0624* Syzygium samarangense KF242094 KF242156 - KF157801 KF254880 KF254859 -
C. tainanense CBS 143666* Capsicum annuum MH728818 MH728823 MH805845 MH781475 MH846558 - -
C. temperatum Coll883* Vaccinium macrocarpon JX145159 - - - JX145211 - -
C. theobromicola ICMP 18649 Theobroma cacao JX010294 JX010006 JX009869 JX009444 JX010447 JX009591 JX010139
C. ti ICMP 4832* Cordyline sp. JX010269 JX009952 JX009898 JX009520 JX010442 JX009649 JX010123
C. tropicale CBS 124949*=ICMP 18653 Theobroma cacao JX010264 JX010007 JX009870 JX009489 JX010407 JX009719 JX010097
C. viniferum GZAAS5.08601* Vitis vinifera JN412804 JN412798 - JN412795 JN412813 - -
C. wuxiense CGMCC 3.17894* Camellia sinensis KU251591 KU252045 KU251939 KU251672 KU252200 - KU252101
C. xanthorrhoeae BRIP 45094*=ICMP 17903 = CBS 127831 Xanthorrhoea preissii JX010261 JX009927 JX009823 JX009478 JX010448 JX009653 JX010138
C. yulongense CFCC 50818* Vaccinium dunalianum MH751507 MK108986 MH793605 MH777394 MK108987 MH793604 MK108988
Colletotrichum sp. BRIP 58074a Citrus australasica MK469999 MK470017 MW091975 MK470089 MK470053 - MK470035

Phylogenetic analyses

Novel sequences were generated from the nine strains in this study, and all reference available sequences of Colletotrichum species were downloaded from GenBank. Multiple sequence alignments for ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS were constructed and carried out using the MAFFT v.7.11 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh et al. 2019) with the default settings, and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted individually for each locus and then as combined analyses of seven loci (ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian.

Inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) using RaxML-HPC2 on XSEDE (8.2.12) (Miller et al. 2012; Stamatakis 2014) and MrBayes on XSEDE (3.2.7a), respectively (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012). For ML analyses the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included seven parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 1000 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 2).

Results

Phylogenetic analyses

Nine strains of Colletotrichum isolated from leaves of Rosa chinensis and fruit of Juglans regia in Mengyin County, Shandong Province, China, were grown in culture. Among the nine Colletotrichum isolates were identified a new species and two known species based on an analysis of combined ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS gene sequences composed of 69 isolates of C. gloeosporioides species complex and C. boninense (CBS 123755) as the outgroup taxon.

A total of 3953 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1–619, GAPDH: 620–929, CHS-1: 930–1229, ACT: 1230–1542, TUB2: 1543–2288, CAL: 2289–3028, GS: 3029–3953. Of these characters, 2667 were constant, 674 were variable and parsimony-uninformative, and 612 were parsimony-informative.

The Bayesian analysis lasted 4,685,000 generations, resulting in 4686 total trees, of which 3515 trees were used to calculate the posterior probabilities. The BI posterior probabilities were plotted on the ML tree. For the BI and ML analyses, HKY+G for GAPDH and ACT, SYM+I+G for ITS, K80+I+G for CHS-1, GTR+G for GS and CAL, HKY+I for TUB2 were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, the ML tree is presented (Fig. 1).

Figure 1. 

Phylogram of Colletotrichum gloeosporioides complex based on combined ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS genes. The ML and BI bootstrap support values above 50% and 0.90 BYPP are shown at the first and second position, respectively. Strains marked with “*” are ex-type or ex-epitype. Strains from this study are shown in red. Two branches were shortened to fit the page size-these are indicated by the symbol (//) with an indication number showing how many times they are shortened.

ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.90) are shown as first and second position above nodes, respectively. The 70 strains were assigned to 60 species clades based on the seven gene loci phylogeny (Fig. 1). The nine strains studied here represented a novel species and two known species. The new species of C. mengyinense showed a close relationship to C. fructicola (MFLU 090228) with full support (ML-BS: 100% and BYPP: 1). The strains SAUCC200952, SAUCC200954 and SAUCC201001 belong to C. gloeosporioides (IMI356878) with full support (ML-BS: 100% and BYPP: 1) by the multi-locus phylogeny. The strains SAUCC200204 and SAUCC201152 belong to C. pandanicola (MFLU 18-0003) with good support (ML-BS: 94% and BYPP: 0.99) by the multi-locus phylogeny.

Taxonomy

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., Atti Reale Ist. Veneto Sci. Lett. Arti., ser. 6, 2: 670. 1884

Figure 2

Vermicudaria gloeosporioides Penz., Michelia 2: 450, 1882. Basionym.

Description

Lesion fruit, round or irregular, dark brown slightly sunken center, brown at margin. Asexual morph developed on PDA. A mass of orange conidia grows in the white mycelium of PDA after 14 days in light at 25 °C. Conidia, hyaline, smooth-walled, subcylindrical, both ends round, 1–3-guttulate, contents granular. Conidia on PDA (10.6–16.5 × 4.3–5.3 µm, mean ± SD = 14.9 ± 1.5 × 4.9 ± 0.3 μm, L/W ratio = 3.0, n = 40). Sexual morph not observed. Conidiogenous cells subcylindrical, straight to curved, 4.7–12.7 × 3.1–4.0 µm, opening 1.5–2.0 μm diam. Conidiophores hyaline, smooth walled, septate, branched.

Figure 2. 

Colletotrichum gloeosporioides (SAUCC201001) a lesion fruit of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata e conidiophores, conidiogenous cells and conidia f–h conidia. Scale bars: 10 μm (e–h).

Culture characteristics

Colonies on PDA flat with entire margin, aerial mycelium white, floccose cottony; surface and reverse grayish in the center and white margin. PDA attaining max 81 mm in diameter after 7 days, at 25 °C, growth rate 8.7–11.5 mm/day. Colonies on SNA sparse hyphae, slow growth.

Specimens examined

China, Shandong Province: Mengyin County, Mengshan, on diseased fruit of Juglans regia, 25 July 2020, T.C. Mu, paratype HSAUP200952, ex-paratype living culture SAUCC200952. China, Shandong Province: Mengyin County, Mengshan, on diseased fruit of Juglans regia, 25 July 2020, T.C. Mu, paratype HSAUP200954, ex-paratype living culture SAUCC200954. China, Shandong Province: Mengyin County, Mengshan, on diseased fruit of Juglans regia 25 July 2020, T.C. Mu, paratype HSAUP201001, ex-paratype living culture SAUCC201001.

Notes

Colletotrichum gloeosporioides was originally described as Vermicularia gloeosporioides on fruit of Citrus sinensis in Italy and this species placed in Colletotrichum by Corda (Weir et al. 2012; Cannon et al. 2008). In the present study, three strains (SAUCC200952, SAUCC200954 and SAUCC201001) are clustered to C. gloeosporioides clade in the combined phylogenetic tree (Fig. 1). Morphologically, our strains were similar to C. gloeosporioides by conidia (10.6–16.5 × 4.3–5.3 vs. 12.0–17.0 (–23.5) × 4.5–6.0 μm, mean:14.9 × 4.9 vs. 14.4 × 5.6 μm). We therefore consider the isolated strain as C. gloeosporioides.

Colletotrichum mengyinense T.C. Mu, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 841265
Figure 3

Etymology

Named after Mengyin County where the fungus was collected.

Diagnosis

Colletotrichum mengyinense can be distinguished from the phylogenetically most closely related species C. fructicola (MFLU 090228) by its large conidia (12.5–15.7 × 4.8–6.1 vs. 9.7–14.0 × 3.0–4.3 μm), and five loci (2/509 in the ITS region, 1/139 GAPDH, 9/237 ACT, 8/410 TUB2 and 20/727 GS).

Type

China, Shandong Province: Mengyin County, on diseased leaves of Rosa chinensis, 25 July 2020, T.C. Mu, holotype HSAUP200702, ex-type living culture SAUCC200702.

Description

Leaf spots discoid to irregular, brown or tanned. Asexual morph developed on SNA. A yellowish or orange mass appearing just as accumulations of conidia on the surface of the medium of SNA after 14 days in light at 25 °C. Conidia one-celled, hyaline, smooth-walled, subcylindrical, both ends round, contents granular. Conidia on SNA (12.5–15.7 × 4.8–6.1 µm, mean ± SD = 14.3 ± 1.1 × 5.3 ± 0.4 μm, L/W ratio = 2.7, n = 40). Sexual morph not observed. Conidiogenous cells subcylindrical, hyaline, 5.3–15.5 × 2.9–4.9 μm, opening 1.7–2.5 μm diam. Conidiophores hyaline, smooth walled, septate, branched.

Figure 3. 

Colletotrichum mengyinense (SAUCC200702) a branch with leaves of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata e-g conidiophores, conidiogenous cells and conidia h–j conidia. Scale bars: 10 μm (e–j).

Culture characteristics

Colonies on PDA flat with entire margin, aerial mycelium white or gray, floccose cottony; surface and reverse gray in the center and grayish margin. PDA attaining 69.3–75.6 mm in diameter after 7 days, at 25 °C, growth rate 9.9–10.8 mm/day. Colonies on SNA sparse hyphae, slow growth.

Additional specimen examined

China, Shandong Province: Mengyin County, on diseased fruit of Juglans regia, 25 July 2020, T.C. Mu, paratype HSAUP200912, ex-paratype living culture SAUCC200912. China, Shandong Province: Mengyin County, on diseased fruit of Juglans regia, 25 July 2020, T.C. Mu, paratype HSAUP200913, ex-paratype living culture SAUCC200913. China, Shandong Province: Mengyin County, on diseased fruit of Juglans regia, 25 July 2020, T.C. Mu, paratype HSAUP200983, ex-paratype living culture SAUCC200983.

Notes

Phylogenetic analysis of a combined seven gene showed that Colletotrichum mengyinense formed an independent clade (Fig. 1) and is phylogenetically distinct from C. fructicola (Prihastuti et al. 2009). This species can be distinguished from C. fructicola by 40 different nucleotides (2/509 in the ITS region, 1/139 in the GAPDH region, 9/237 ACT, 8/410 TUB2 and 20/727 GS). What’s more, C. mengyinense differs from C. fructicola in having large conidia (12.5–15.7 × 4.8–6.1 vs. 9.7–14.0 × 3.0–4.3 μm, mean: 14.3 × 5.3 vs. 11.53× 3.55 μm). Therefore, we establish this fungus as a novel species.

Colletotrichum pandanicola Tibpromma & K.D. Hyde, MycoKeys 33:47. (2018)

Figure 4

Description

Lesion fruit, round or irregular, dark brown slightly sunken center, brown at margin. Asexual morph developed on SNA. A mass of yellowish or orange creamy conidial droplets at the inoculum point on SNA after 14 days in light at 25 °C. Born in conidiomata, conidia first take an ovoid shape, then become subcylindrical with rounded ends, contents granular. Conidia on SNA (14.2–17.9 × 4.6–6.0 µm, mean ± SD = 16.1 ± 0.9 × 5.4 ± 0.3 μm, L/W ratio = 2.9, n = 40). Sexual morph not observed. Conidiogenous cells subcylindrical, hyaline, 5.5–23.9 × 2.6–6.3 μm, opening 1.1–1.5 μm diam. Conidiophores branched, hyaline, smooth walled, septate, some septa disappeared at the end, contents granular.

Figure 4. 

Colletotrichum pandanicola (SAUCC201152) a lesion fruit of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 7 days on PDA d conidiomata e, f conidiophores, conidiogenous cells and conidia g, h conidiophores, conidiogenous cells i–k conidia. Scale bars: 10 μm (e–k).

Culture characteristics

Colonies on PDA flat with entire margin, aerial mycelium white, floccose cottony; light gray in the center and pale white margin, reverse white to pale brownish. PDA attaining 58.1–82.6 mm in diameter after 7 days, at 25 °C, growth rate 8.3–11.8 mm/day. Colonies on SNA sparse hyphae, slow growth.

Specimens examined

China, Shandong Province: Mengyin County, Mengshan, on diseased fruit of Juglans regia. 25 July 2020, T.C. Mu, paratype HSAUP200204, ex-paratype living culture SAUCC200204. China, Shandong Province: Mengyin County, Mengshan, on diseased fruit of Juglans regia. 25 July 2020, T.C. Mu, paratype HSAUP201152, ex-paratype living culture SAUCC201152.

Notes

Colletotrichum pandanicola was originally described from the healthy leaves of Pandanus sp. (MFLU 18-0003, Pandanaceae) in Thailand (Tibpromma et al. 2018). In the present study, two strains (SAUCC200204 and SAUCC201152) are clustered to the C. pandanicola clade in the combined phylogenetic tree (Fig. 1). Morphologically, our strains were similar to C. pandanicola by conidia (14.2–17.9 × 4.6–6.0 vs. 9.0–18.0 × 4.0–8.0 μm, mean:16.1 × 5.4 vs. 13.39 × 5.35 μm). We therefore consider the isolated strains as C. pandanicola.

Discussion

In this study, the Colletotrichum specimens of diseased leaves and fruits were collected in Mengyin, Shandong Province, China. A temperate monsoon climate and an abundance of fruit trees provide the proper conditions for anthracnose propagation. As a result, 70 reference sequences (including an outgroup taxon: C. boninense CBS 123755) were selected based on BLAST searches of NCBI’s GenBank nucleotide database and were included in the phylogenetic analyses (Table 2).

Phylogenetic analyses based on seven combined loci (ITS, GAPDH, CHS-1, ACT, TUB2, CAL and GS), as well as morphological characters of the asexual morph obtained in culture, were contributed to knowledge of the diversity of Colletotrichum species in Shandong Province. Based on a large set of freshly collected specimens from Shandong province, China, nine strains of Colletotrichum species were isolated from two host genera (Table 2). A new species is proposed: C. mengyinense. In a previous report, C. gloeosporioides has been isolated from Juglans regia (Zhu et al. 2014). Colletotrichum pandanicola was described from Pandanus sp. (Pandanaceae) in Thailand (Tibpromma et al. 2018) and C. pandanicola is first reported from Juglans regia in China. In this study, we described and illustrated C. gloeosporioides and C. pandanicola again.

Previously, species identification of Colletotrichum was largely referred to the host-specificity and pure culture characteristics, leading to the chaos of names (Weir et al. 2012). On the other hand, based on a polyphasic approach and known morphology, more than one species of Colletotrichum can colonize a single host, while one species can be associated with different hosts (Damm et al. 2012). It revealed diversity of Colletotrichum species from different hosts. Our study supported this result. For example, C. pandanicola (SAUCC200204 and SAUCC201152) and C. gloeosporioides (SAUCC200952, SAUCC200954 and SAUCC201001) were collected from Juglans regia. In addition, isolates of C. mengyinense were obtained from two hosts (Juglans regia and Rosa chinensis). The morphological descriptions and molecular data for species of Colletotrichum represent an important resource and basis for plant pathologists and fungus taxonomists.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 31900014, 31750001 and 31770016).

References

  • Bhunjun CS, Phukhamsakda C, Jayawardena RS, Jeewon R, Promputtha I, Hyde KD (2021) Investigating species boundaries in Colletotrichum. Fungal Diversity 107: 107–127. https://doi.org/10.1007/s13225-021-00471-z
  • Cai L, Hyde KD, Taylor PWJ, Weir BS, Waller JM, Abang MM, Zhang ZJ, Yang YL, Phoulivong S, Liu ZY, Prihastuti H, Shivas RG, McKenzie EHC, Johnston PR (2009) A polyphasic approach for studying Colletotrichum. Fungal Diversity 39: 183–204.
  • Cannon PF, Buddie AG, Bridge PD (2008) The typification of Colletotrichum gloeosporioides. Mycotaxon 104: 189–204.
  • Cannon PF, Damm U, Johnston PR, Weir BS (2012) Colletotrichum - current status and future directions. Studies in Mycology 73: 181–213. https://doi.org/10.3114/SIM0014
  • Chomnunti P, Hongsanan S, Aguirre-Hudson B, Tian Q, Peršoh D, Dhami MK, Alias AS, Xu J, Liu X, Stadler M, Hyde KD (2014) The sooty moulds. Fungal Diversity 66: 1–36. https://doi.org/10.1007/S13225-014-0278-5
  • Corda ACI (1831) Die Pilze Deutschlands. In: Sturm J (Ed.) Deutschlands Flora in Abbildungen nach der Natur mit Beschreibungen. Sturm, Nürnberg 3(12), 33–64.
  • Damm U, Cannon PF, Woudenberg JH, Johnston PR, Weir BS, Tan YP, Shivas RG, Crous PW (2012) The Colletotrichum boninense species complex. Studies in Mycology 73: 1–36. https://doi.org/10.3114/sim0002
  • Damm U, Woudenberg JHC, Cannon PF, Crous PW (2009) Colletotrichum species with curved conidia from herbaceous hosts. Fungal Diversity 39: 45–87.
  • Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The Top 10 fungal pathogens in molecular plant pathology. Molecular Plant Pathology 13: 414–430. https://doi.org/10.1111/J.1364-3703.2011.00783.X
  • Jayawardena RS, Hyde KD, Jeewon R, Li XH, Liu M, Yan JY (2016) Mycosphere Essay 6: Why is it important to correctly name Colletotrichum species? Mycosphere 7: 1076–1092. https://doi.org/10.5943/mycosphere/si/2c/1
  • Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20: 1160–1166. https://doi.org/10.1093/bib/bbx108
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/MOLBEV/MSW054
  • Liu F, Weir BS, Damm U, Crous PW, Wang Y, Liu B, Wang M, Zhang M, Cai L (2015) Unravelling Colletotrichum species associated with Camellia: employing ApMat and GS loci to resolve species in the C. gloeosporioides complex. Persoonia 35: 63–86. https://doi.org/10.3767/003158515X687597
  • Miller MA, Pfeiffer W, Schwartz T (2012) The CIPRES science gateway: enabling high-impact science for phylogenetics researchers with limited resources. Proceedings of the 1st Conference of the Extreme Science and Engineering Discovery Environment. Bridging from the extreme to the campus and beyond. Association for Computing Machinery 39: 1–8. https://doi.org/10.1145/2335755.2335836
  • Nylander JAA (2004) MrModeltest v. 2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
  • Prihastuti H, Cai L, Chen H, Mckenzie EHC, Hyde KD (2009) Characterization of Colletotrichum species associated with coffee berries in northern Thailand. Fungal Diversity 39: 89–109.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029
  • Tibpromma S, Hyde KD, Bhat JD, Mortimer PE, Xu J, Promputtha I, Doilom M, Yang JB, Tang AMC, Karunarathna SC (2018) Identification of endophytic fungi from leaves of Pandanaceae based on their morphotypes and DNA sequence data from southern Thailand. MycoKeys: 25–67. https://doi.org/10.3897/mycokeys.33.23670
  • Weir BS, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115–180. https://doi.org/10.3114/sim0011