First Report of Colletotrichum fructicola, C. rhizophorae sp. nov. and C. thailandica sp. nov. on Mangrove in Thailand

Colletotrichum, a genus within the phylum Ascomycota (Fungi) and family Glomerellaceae are important plant pathogens globally. In this paper, we detail four Colletotrichum species found in mangrove ecosystems. Two new species, Colletotrichum rhizophorae and C. thailandica, and a new host record for Colletotrichum fructicola were identified in Thailand. Colletotrichum tropicale was collected from Taiwan’s mangroves and is a new record for Rhizophora mucronata. These identifications were established through a combination of molecular analysis and morphological characteristics. This expanded dataset for Colletotrichum enhances our understanding of the genetic diversity within this genus and its associations with mangrove ecosystems. The findings outlined herein provide data on our exploration of mangrove pathogens in Asia.


Introduction
Mangroves, the coastal ecosystems where land and sea merge, have been a subject of fascination for ecologists, conservationists, and nature enthusiasts for decades.Thailand has an extensive coastline and is home to numerous mangrove forests that have not only drawn the attention of researchers but have also unveiled a lesser-known yet incredibly diverse facet of these ecosystems-the extraordinary diversity of fungi they harbor [1,2].This introduction sets the stage for the exploration of the captivating world of Thai mangroves and their rich fungal diversity.Thailand's mangrove forests are a critical component of its coastal biodiversity and ecological integrity.They serve as a protective buffer against erosion, tidal surges, provide invaluable breeding grounds for marine species, and contribute significantly to carbon sequestration and climate change mitigation [3].While the spotlight is often on the charismatic fauna and flora within mangroves, the fungi inhabiting these environments have, until recently, remained less studied.Recent studies have shed light on the remarkable diversity of fungi in Thai mangroves [4][5][6][7].These fungi exhibit unique adaptations to the harsh conditions of mangrove ecosystems, thriving in saline environments and forming intricate relationships with mangrove trees and other microorganisms [1,2].They play pivotal roles in nutrient cycling, organic matter decomposition, and symbiotic associations, all of which are essential for the health and sustainability of mangrove ecosystems [8,9].
In this study, we studied the mangroves of Thailand and Taiwan to uncover the phylogenetic diversity of Colletotrichum species associated with Rhizophora apiculata and R. mucronata, respectively.The aim of this study was to identify these isolates based on phylogenetic data and morphology to confirm their novel associations in mangrove ecosystems.

Sampling and Examination of Specimens
Fresh leaf samples were collected in 2017 from Rhizophora apiculata in Thailand.Fresh specimens were taken to the laboratory in paper bags, examined, and described.Morphological characters of conidiomata were examined using an Olympus SZX16 stereo microscope (Olympus Corporation, Tokyo, Japan).Micromorphology was studied and photographed using a Nikon Eclipse Ni compound microscope with a Microscope Camera DS-Ri2 (Nikon Corporation, Tokyo, Japan).All image measurements were made with the Image Frame Work program v. 0.9.7 (Tarosoft ®, Nontha Buri, Thailand).Photoplates were made using Adobe Photoshop CC 2019 version 20.0.1 (Adobe Systems, San Jose, CA, USA).
The cultures were acquired using the tissue isolation technique as described in the study of Norphanphoun et al. [37].Single hyphal tips were transferred onto 2% potato agar (PA) plates at room temperature (25 • C ± 2) throughout a one-week period: 12 hours dark and 12 hours light.The cultural features were observed and documented at intervals of 5, 7, and 14 days.The morphological characteristics of the culture were analyzed during the entire cultivation duration.In order to conduct further experiments, pure cultures were cultivated on potato dextrose agar (PDA) (HiMedia Laboratories LLC, Kennett Square, PA, USA).Dried and living cultures were deposited in the culture collection at Mae Fah Luang University (MFLUCC) and herbarium collection (MFLU), Chiang Rai, Thailand.The enumeration of Faces of Fungi (https://www.facesoffungi.org/) was conducted following the methodology outlined in Jayasiri et al. [38].
The amplification reactions were carried out using the following protocol: 25 µL reaction volume containing 1 µL of DNA template, 1 µL (20 µM stock concentration) of each forward and reverse primers, 12.5 µL of DreamTaq Green PCR Master Mix (2×) (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 9.5 µL of double-distilled water (ddH 2 O).The PCR thermal cycling program for each locus is described in Table 1.PCR products were analyzed using 1.7% TAE agarose gels containing the 100 bp DNA Ladder RTU (Bio-Helix Co., Ltd., Taipei, Taiwan) to confirm the presence of amplicons at the expected molecular weight.The purification and sequencing of PCR products using the amplification primers specified above were conducted at SolGent Co., Ltd., located in Daejeon, Republic of Korea.

Phylogenetic Analysis
The raw readings were processed and organized into contigs using Geneious Prime ® 2023.2.1 Java Version 11.0.18+10(64-bit) software (Biomatters Inc., Boston, MA, USA).The newly generated sequences were utilized as queries to conduct a BLASTn search against the nonredundant (nr) database in GenBank (https://www.ncbi.nlm.nih.gov/;accessed on 1 September 2023).The retrieval of similar sequences was conducted, followed by the construction of numerous alignments.The GenBank taxonomy browser was utilized to verify all sequences classified as Colletotrichum in the database.BioEdit version 7.2.5 (Ibis Biosciences, Carlsbad, CA, USA) [43] was used to assign open reading frames of the protein coding sequences of actin, gapdh, β-tubulin, chs-1, and cal according to reference sequences in the GenBank database.The combined sequence data of all loci were used to perform maximum likelihood (ML) and Bayesian inference analysis (BI).The dataset consisted of 126 taxa of the Colletotrichum gloeosporioides species complex and two taxa from singleton species as outgroups, C. arecacearum strains MH0003 and MH0003-1.Outgroup sequences were selected based on preliminary analysis of the multigene phylogeny of the Colletotrichum species complex dataset.All taxa used for these analyses can be found in Table 2.
Sequences were aligned for each locus separately using the MAFFT v.7.110 online program (http://mafft.cbrc.jp/alignment/server/;accessed on 19 September 2023) [44].TrimAl/readAl v1.2.program was used to trim ambiguously aligned positions [45].The software BioEdit version 7.2.5 was utilized to make additional manual edits as needed [43].The congruency of genes and their potential for combination were assessed using a partition homogeneity test (PHT) conducted using PAUP* 4.0b10 software [46].The concatenated sequence alignments were acquired from MEGA version 7.0.14 and version 10.1.0,as reported by Kumar et al. [47] and Tamura et al. [48], respectively.Geneious Prime ® 2023.2.1 was used to convert file format to Nexus BI analyses.The data were divided into the following categories: ITS, act-exon, gapdh-exon, βtubulin-exon, chs-1-exon, cal-exon, act-intron, gapdh-intron, β-tubulin-intron, and cal-intron.The researchers utilized the software RAxML-HPC2 on XSEDE to conduct maximum likelihood (ML) analysis, which was implemented using the CIPRES Science Gateway web server (https://www.phylo.org/portal2/;accessed on 20 November 2023) [49].A total of 1000 bootstrap repeats were conducted in a swift manner, employing the GTRGAMMA model to simulate nucleotide evolution.The researchers conducted a Bayesian inference analysis by utilizing the Markov Chain Monte Carlo (MCMC) algorithm, which was implemented on the CIPRES Science Gateway web server.Specifically, they used MrBayes on XSEDE, as described by Miller et al. [49].The optimal nucleotide substitution model for each partition was individually calculated using MrModeltest version 2.2 (Boston, MA, USA), as shown in Table 3 [50].The computation of posterior probability involved the execution of two independent runs, each consisting of four chains.These runs were initiated from a randomly generated tree topology.A total of 10 million generations were executed for the given dataset.The sampling of trees occurred at regular intervals of 100 generations.According to Ronquist et al. [51], a quarter of the trees were excluded as burn-in values, while the average standard deviation of split frequencies reached convergence below 0.01.

Gene
Substitution Model The phylogram was generated using FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/ figtree/) [52], a software tool commonly used for visualizing phylogenetic trees.The final figure was created using Adobe Illustrator CC version 23.0.1 (64-bit) and Adobe Photoshop CC version 20.0.1 release, both products developed by Adobe Systems in California, USA.The newly produced sequences in this investigation were deposited in GenBank as indicated in Table 2.The completed alignments and trees were submitted to TreeBASE.
The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model with a pairwise homoplasy index (PHI) test was used to analyze the newly generated taxon and its most phylogenetically close neighbors [53].The PHI test was performed in SplitsTree v. 4.14.6 [54,55] with a five-locus concatenated dataset (ITS, act, gapdh, β-tubulin, chs-1, and cal) to determine the recombination level among phylogenetically closely related species.A pairwise homoplasy index below a 0.05 threshold (Φw < 0.05) indicated the presence of significant recombination in the dataset.The relationship between closely related species was visualized by constructing a split graph.

Results
The results of the partition homogeneity test (PHT) for the phylogenetic tree were not significant (95% level), which suggests that the individual datasets can be combined.To assess tree topology and clade support, single-locus phylogenetic trees were also generated before the combined gene tree was conducted.In this research, we introduce two novel Colletotrichum species alongside two known species.
The analysis of six genetic loci using both maximum likelihood (ML) and Bayesian inference (BI) methods resulted in a phylogenetic tree with well-supported clades, as shown in Figure 1.Within this study, we propose the recognition of two novel species, namely C. rhizophorae and C. thailandica, with robust statistical backing, signified by a high bootstrap support of 95% (BSML) and a posterior probability of 0.85 (PPBI).In terms of known species, two strains originating from mangrove habitats in Thailand (MFLUCC 17-1752 and MFLUCC 17-1753) were classified as members of the species C. fructicola, while a strain from Taiwan (NTUCC) was identified as C. tropicale.Notably, MFLUCC 17-1752 and MFLUCC 17-1753 clustered within the C. fructicola species group with substantial support: a 98% bootstrap support (BSML) and a posterior probability of 1.00 (PPBI).On the other hand, strain NTUCC was grouped within the C. tropicale species cluster, exhibiting a strong 99% bootstrap support (BSML) and a posterior probability of 1.00 (PPBI).It is noteworthy that all newly introduced strains in this study shared the same topological arrangement as the preliminary analysis of the Colletotrichum species complex.
To assess evolutionary independence, we employed the GCPSR concept on our strain dataset and its closely related taxa.The pairwise homoplasy index (PHI or Φw) is a crucial metric, and a value below 0.05 suggests the presence of substantial genetic recombination within a dataset.Figure 2 shows that our GCPSR analysis gave a PHI of 0.3688 for all closely related taxa in this study.This means that there was no significant genetic mixing between these strains and their sister taxa.Since we saw that the newly introduced species were very different from each other in terms of their phylogeny, we extended the GCPSR analysis to isolate only these new species.The results showed that the PHI value was greater than 0.05 (Φw = 1.0) for both newly taxon C. rhizophorae and C. thailandica isolates with the known species C. pandanicola.This clearly shows that these two new species have not been recombined in a significant way.This substantiates the distinct species status of all these isolates.Culture characteristics: Colonies on CMA reaching 7-8 cm diam after 7 d at room temperature (±25 °C), under light 12 h/dark 12 h, colonies rhizoid to filamentous, dense, flat or raised surface, with filiform margin, white from above and white to pale-yellow reverse, with producing grouped pycnidia.Colonies on WA with sterilized sticks, reaching 5 cm diam after 7 d at room temperature (±25 °C), under light 12 h/dark 12 h, colonies rhizoid to filamentous, dense, flat surface, with filiform margin, dark green from above and reverse, with producing pycnidia on sticks and immersed pycnidia under media.
Culture characteristics: Colonies on PDA reaching 6-7 cm diam after 7 d at room temperature (±25 • C), under light 12 h/dark 12 h, colonies filamentous to circular, medium dense, aerial mycelium on surface flat, with irregular margin, white from above and reverse, with producing pycnidia and yellow spore mass  Notes: We introduce Colletotrichum rhizophorae as a novel species discovered within Rhizophora apiculata, a mangrove plant in Thailand (Figure 5).This classification is supported by morphological and phylogenetic evidence, as depicted in Figure 1.The phylogenetic analysis demonstrates that this new taxon closely associates with C. thailandica (Figure 1).However, notable distinctions in morphology are observed between C. rhizophorae and C. thailandica, particularly in conidia, conidiophores, and conidiogenous cells (refer to Figures 5 and 6).In order to establish evolutionary independence, we applied the GCPSR concept to C. rhizophorae and its neighboring taxa.Our dataset yielded a PHI value Notes: We introduce Colletotrichum rhizophorae as a novel species discovered within Rhizophora apiculata, a mangrove plant in Thailand (Figure 5).This classification is supported by morphological and phylogenetic evidence, as depicted in Figure 1.The phylogenetic analysis demonstrates that this new taxon closely associates with C. thailandica (Figure 1).However, notable distinctions in morphology are observed between C. rhizophorae and C. thailandica, par-ticularly in conidia, conidiophores, and conidiogenous cells (refer to Figures 5 and 6).In order to establish evolutionary independence, we applied the GCPSR concept to C. rhizophorae and its neighboring taxa.Our dataset yielded a PHI value exceeding 0.05 (Φw = 0.363), indicating the absence of significant genetic recombination between C. rhizophorae and its sister taxa, namely C. pandanicola and C. thailandica (Figure 2).Furthermore, a comparison of nucleotide sequences within ITS, act, gapdh, β-tubulin, chs-1, and SCDgle revealed discrepancies between C. thailandica and C. rhizophorae (ITS 5 bp, act 3 bp, gapdh 4 bp, β-tubulin 2 bp, chs-1 6 bp, and SCDgle 4 bp).3) µm (mean ± SD = 14.7 ± 1.2 × 4 ± 0.3 µm), hyaline, aseptate, smooth-walled, clavate to cylindrical, one end rounded and one end acute or both ends rounded, guttulate, granular.
Culture characteristics: Colonies on PDA reaching 7-8 cm diam after 10 d at room temperature (±25 • C), under light 12 h/dark 12 h, colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, with filiform margin (curled margin), fluffy, white from above and white to pale-yellow reverse, with producing pycnidia and yellow spore mass.
Notes: Thailand, Wan Yao, Khlung, Chanthaburi, asymptomatic leaf of Rhizophora apiculata, 25 April 2017, Kevin D. Hyde WYKE07AL (dried culture MFLU 23-0480, holotype), living cultures, MFLUCC 17-1924.Notes: Colletotrichum thailandica is introduced here as a new species in the gloeosporioides species complex, a classification supported by both morphological (Figure 6) and phylogenetic data.The phylogenetic analysis underscores the distinctiveness of this new taxon, clearly separating it from other recognized Colletotrichum species (Figure 1).
In order to assess evolutionary autonomy, we applied the GCPSR concept to C. thailandica and its closely related taxa.Our data showed that the PHI value was higher than 0.05 (Φw = 0.363), which means that there was not much genetic mixing between C. thailandica and its closest relatives, C. pandanicola and C. rhizophorae (Figure 2).Since there was a lot of phylogenetic diversity between newly introduced species and species that had already been published, like C. pandanicola, we used GCPSR analysis on a larger dataset.The outcome revealed a PHI value surpassing 0.05 (Φw = 1.0), unequivocally indicating the absence of significant recombination for this new species.As a result, we formally introduce C. thailandica as a distinct species, isolated from Rhizophora apiculata in Thailand.Culture characteristics: Colonies on PDA reaching 7-8 cm diam after 14 d at room temperature (±25 • C), under light 12 h/dark 12 h, colonies filamentous to circular, medium dense, aerial mycelium on surface flat or raised, with filiform margin (curled margin), fluffy, gravy from above and dark gravy reverse, with producing pycnidia and yellow spore mass (Figure 3D).

Discussion
Colletotrichum is a pathogenic genus that affects various plant species, including mangroves [28][29][30].It causes anthracnose, a common disease characterized by dark lesions on leaves, stems, and fruits [16].Several studies have investigated the prevalence and impact of Colletotrichum on mangroves, providing valuable data for understanding its ecology and management strategies [28,35,36].In this study, we focused on the examination of six strains isolated from mangrove ecosystems.Five of these strains were isolated from Rhizophora apiculata in Thailand's mangroves, while one strain originated from Rhizophora mucronata in Taiwan.Among the isolates, Colletotrichum fructicola (MFLUCC 17-1752) was obtained from leaf spot symptoms, while the remaining strains were isolated from asymptomatic leaves.It is important to note that C. fructicola has been found to play different roles in the environment, including as an epiphyte, an endophyte, and a pathogen in a wide range of host species [203].This suggests that the presence of Colletotrichum species in mangrove ecosystems may be more diverse than initially anticipated.These taxa can exhibit various ecological interactions, including their colonization of asymptomatic leaves.As a result, there is potential for the discovery of additional fungal species within mangrove forest zones.These newly discovered species could encompass both those commonly found in other plant species and entirely novel fungal types.
This comprehensive study employed phylogenetic analysis, morphological characterization, and the Genetic Clade-Phenetic Species Recognition (GCPSR) concept to elucidate the taxonomy and evolutionary relationships of these Colletotrichum species within the gloeosporioides species complex according to the guidelines of Chethana et al. [226] and Maharachchikumbura et al. [227].Previously, Weir et al. [42] documented the efficacy of individual genes in discerning species within the gloeosporioides species complex.The study identified the designated barcoding gene for fungi in the gloeosporioides complex, encompassing eight genes: the internal transcribed spacer region (ITS), actin (act), glyceraldehyde-3-phosphate dehydrogenase region (gapdh), beta-tubulin (β-tubulin), chitin synthase (chs-1), calmodulin (cal), glutamine synthetase (GS), and manganese-superoxide dismutase (SOD2).However, it was observed that these genes do not consistently provide a conclusive resolution of relationships for all species within this particular species complex.In the context of C. siamense, the performance of individual genes that can distinguish species within the C. gloeosporioides species complex is notably achieved by examining cal and β-tubulin sequences.Conversely, for C. tropicale, the distinguishing genes encompass β-tubulin, act, GS, and SOD2.In the case of C. fructicola, the pertinent genes for effective differentiation are cal, chs-1, GS, and SOD2.To overcome limitations associated with gene function in species delimitation and to achieve precise identification of Colletotrichum isolates in the present study, a comprehensive approach employing six gene sequences (ITS, act, gapdh, β-tubulin, chs-1, and cal), encompassing 126 strains, and 2 singleton strains as outgroups were used to facilitate the identification of two novel species and to document a new host record from Thailand.Moreover, there is a new record of C. tropicale from Taiwan, using act, β-tubulin, and chs-1.The study encompasses multiple reference isolates of C. fructicola, C. siamense, and C. tropicale.The results of a multigene phylogenetic analysis demonstrated that the combined use of ITS, act, gapdh, β-tubulin, chs-1, and cal offered superior resolution in determining Colletotrichum species, surpassing the efficacy of single-gene analysis.This finding aligns with prior studies conducted by Prihastuti et al. [201] and Weir et al. [42].The results provided valuable insights into the diversity and classification of Colletotrichum species.The phylogenetic analysis, utilizing both maximum likelihood (ML) and Bayesian inference (BI) methods, revealed a well-supported clustering of the new strains within the gloeosporioides species complex clade, alongside sequences previously identified as members of this complex.The robust statistical support, with 100% bootstrap support (BSML) and a posterior probability of 1.00 (PPBI), underlined the validity of the species complex classification (Figure 1).Within this complex, two novel species are formally recognized: C. rhizophorae and C. thailandica.These designations were supported by a high bootstrap support of 99% (BSML) and a posterior probability of 1.00 (PPBI), reaffirming their distinct species status.Additionally, known species, including C. fructicola and C. tropicale, were identified and validated based on their placement within the phylogenetic tree.The application of the GCPSR concept further corroborated the evolutionary independence of these species.The pairwise homoplasy index (PHI or Φw) values exceeding 0.05 indicated a lack of significant genetic recombination within the dataset, highlighting the distinctiveness of the newly proposed species.This was particularly evident in the case of C. rhizophorae and C. thailandica, as their PHI values exceeded 0.05 even when analyzed with closely related taxa.The study delved into the taxonomy of two Colletotrichum species, C. fructicola and C. tropicale, offering significant insights into their classification, morphology, and distribution.
Colletotrichum fructicola, originally described in 2009 from Coffea arabica in Thailand [201], was the subject of taxonomic reevaluation.The study consistently found that the strain under investigation clustered closely with known C. fructicola strains within the gloeosporioides species complex.This clustering was observed both in the preliminary analysis and the final phylogenetic tree, reaffirming its placement within this species complex.Furthermore, morphological similarities, including conidia size, asci size, and ascospore features, provided additional support for the classification of the strain as C. fructicola.Importantly, this study marked a significant milestone in scientific discovery by documenting the first-ever instance of an endophytic fungus isolated from R. apiculata in Thailand.
Colletotrichum tropicale, initially documented from T. cacao leaves in Panama [206], was also investigated in this study.The research employed phylogenetic analysis and examination of conidia morphology to validate the classification of the study's isolate as C. tropicale.This confirmation represented a notable scientific contribution, as it marked the first documented instance of an endophytic fungus isolated from R. mucronata in Taiwan.
These records expand our knowledge of the geographic distribution of these fungal species.In conclusion, this research enhances our understanding of fungal diversity

Figure 2 .
Figure 2. The results of the pairwise homoplasy index (PHI) test for closely related species of Colletotrichum stains in this study using both LogDet transformation and splits decomposition.PHI test results (Φw) > 0.05 indicate no significant recombination within the dataset.

Figure 2 .
Figure 2. The results of the pairwise homoplasy index (PHI) test for closely related species of Colletotrichum stains in this study using both LogDet transformation and splits decomposition.PHI test results (Φw) > 0.05 indicate no significant recombination within the dataset.

Table 1 .
Polymerase chain reaction (PCR) thermal cycling programs for each locus.C for 5 min, 40 cycles of D at 95 • C for 45 s, A at 53 • C for 45 s, E at 72 • C for 2 min, FE at 72 • C for 10 min • C for 30 s, E at 72 • C for 45 s, FE at 72

Table 2 .
Cont. -Queensland Plant Pathology Herbarium; CBS-CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CFCC-China Forestry Culture Collection Center; CGMCC-China General Microbiological Culture Collection Center; ICMP-International Collection of Microorganisms from Plants; IMI-International Mycological Institute; MFLUCC-Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; NTUCC-the Department of Plant Pathology and Microbiology, National Taiwan University Culture Collection.T Ex-type strains.Strains in this study are in bold. BRIP

Table 3 .
The best-fit nucleotide substitution model for each dataset, selected by AIC in MrModeltest.2.2.