Diversity, structure, and synteny of the cutinase gene of Colletotrichum species

Abstract Colletotrichum species complexes are among the top 10 economically important fungal plant pathogens worldwide because they can infect climacteric and nonclimacteric fruit at the pre and/or postharvest stages. C. truncatum is the major pathogen responsible for anthracnose of green and red bell pepper fruit worldwide. C. brevisporum was recently reported to be a minor pathogen of red bell pepper fruit in Trinidad, but has recently been reported as pathogenic to other host species in other countries. The ability of these phytopathogens to produce and secrete cutinase is required for dismantling the cuticle of the host plant and, therefore, crucial to the necrotrophic phase of their infection strategy. In vitro bioassays using different lipid substrates confirmed the ability of C. truncatum and C. brevisporum isolates from green and red bell peppers to secrete cutinase. The diversity, structure and organization and synteny of the cutinase gene were determined among different Colletotrichum species. Cluster analysis indicated a low level of nucleotide variation among C. truncatum sequences. Nucleotide sequences of C. brevisporum were more related to C. truncatum cutinase nucleotide sequences than to C. gloeosporioides. Cluster patterns coincided with haplotype and there was evidence of significant positive selection with no recombination signatures. The structure of the cutinase gene included two exons with one intervening intron and, therefore, one splice variant. Although amino acid sequences were highly conserved among C. truncatum isolates, diversity “hot spots” were revealed when the 66‐amino acid coding region of 200 fungal species was compared. Twenty cutinase orthologues were detected among different fungal species, whose common ancestor is Pezizomycotina and it is purported that these orthologues arose through a single gene duplication event prior to speciation. The cutinase domain was retained both in structure and arrangement among 34 different Colletotrichum species. The order of aligned genomic blocks between species and the arrangement of flanking protein domains were also conserved and shared for those domains immediately located at the N‐ and C‐terminus of the cutinase domain. Among these were an RNA recognition motif, translation elongation factor, signal peptide, pentatricopeptide repeat, and Hsp70 family of chaperone proteins, all of which support the expression of the cutinase gene. The findings of this study are important to understanding the evolution of the cutinase gene in C. truncatum as a key component of the biotrophic–necrotrophic switch which may be useful in developing gene‐targeting strategies to decrease the pathogenic potential of Colletotrichum species.


| INTRODUC TI ON
Many plant pathogenic fungi have complex life cycles which enable them to interact differently with their hosts (Horbach, Navarro-Quesada, Knogge, & Deising, 2011). As such, a two-stage life cycle is typical of hemibiotrophic fungi: the initial biotrophic phase: the fungus grows and survives in a quiescent state to maintain host viability, therefore, the host remains asymptomatic and without host tissue destruction; the second necrotrophic phase: tissue is decomposed through cellular dismantling and destruction, assimilation of the contents of dead or dying cells which results in symptom manifestation and subsequent death of plant tissue (Brunner, Torriani, Croll, Stukenbrock, & McDonald, 2013;Stone, 2001).
Colletotrichum is one of the most economically important genera of plant pathogenic fungi with a membership of more than 200 species, most of which enjoy a broad host range worldwide, and cause anthracnose of fruit and vegetable crops in tropical and subtropical climates (Udayanga, Manamgoda, Liu, Chukeatirote, & Hyde, 2013).
A comparative study involving the genome and transcriptome of C. higginsianum and C. graminicola indicated that (a) both fungal species possessed numerous pathogenicity-related genes, with genetic footprints reminiscent of biotrophic and necrotrophic pathogens, (b) there are differences in the expression profile of these pathogenicity-related genes, and (c) induction of gene expression is highly regulated and stage-specific (Alkan et al., 2015;Damm, O'Connell, Groenewald, & Crous, 2014;Kleemann et al., 2012;Liu et al., 2013;O'Connell et al., 2012).
The interaction between a hemibiotroph and its plant host is highly specialized both structurally and physiologically such that the duration of the biotrophic or necrotrophic phase differs among hemibiotrophic pathogens and is dependent on physiological triggers of the host plant (Laluk & Mengiste, 2010). During fruit ripening, specific physiological changes occur in the host, including those involved in the natural ripening process, in recognition of the fungal pathogen, and in activation of the host's defense response (Alkan et al., 2015).
Some of these physiological modifications include remodelling of the cell wall architecture (Brummell et al., 1999;Hückelhoven, 2007), accumulation of soluble sugar, altered bioactivity of phytoalexins, and phytoanticipins (Prusky, 1996); changes in the pH of the cytoplasm of the host plant cell (Prusky, Alkan, Mengiste, & Fluhr, 2013); and cuticle biosynthesis (Bargel & Neinhuis, 2005). Alterations of cuticle composition, structure, and deposition are involved in triggering the switch from a quiescent state to a necrotrophic state which in turn, allow fungal pathogens to penetrate the cuticle, infect and cause destruction of fruit tissue (Agudelo-Romero et al., 2015;Alkan et al., 2015;Bhadauria et al., 2013;Blanco-Ulate et al., 2014). These changes are regulated by a complex interplay of hormonal signals which affect both plant defense responses and resistance to pathogen invasion (Giovannoni, 2001;Seymour, Østergaard, Chapman, Knapp, & Martin, 2013;Voisin et al., 2009).
The cuticle is considered to be a "lipidized cell wall region" that is composed of several compounds, with cutin being the most abundant, followed by cuticular waxes which are a combination of organic solvent-soluble, very long-chain fatty acid compounds (Lara, Belge, & Goulao, 2015;Martin & Rose, 2014). As a physical barrier, the cuticle surrounds the epidermis of fruits and serves a number of functions including (a) to protect the plant against the physical, environmental and biological stresses and pathogen invasion, (b) to reduce the effects of internal water loss, and (c) to maintain plant organ integrity by providing mechanical support Chen et al., 2013;Lara, Belge, & Goulao, 2014). In pathogenicity tests, C. truncatum isolates are pathogenic on Capsicum fruits after wounding the fruit surface, and most produced a low level of infection on nonwounded fruit (De Silva et al., 2019;Ramdial & Rampersad, 2015).
In climacteric fruits, changes in cuticle thickness and composition change during ripening (Martin & Rose, 2014). However, in nonclimacteric fruits, like bell pepper, once maximum cutin monomer levels are reached during development, it decreases steadily as the fruit ripens (Kosma et al., 2010). This demonstrates the significance of the cuticle to infection by Colletotrichum spp. (Auyong, Ford, & Taylor, 2015).
The cuticle also has active roles in host defense signalling in addition to local and systemic resistance against a variety of pathogenic fungi. Fungal spores adhere to the surface of the host plant which signals production of low levels of cutinase activity which produces small amounts of cutin monomers (Woloshuk & evolution of the cutinase gene in C. truncatum as a key component of the biotrophicnecrotrophic switch which may be useful in developing gene-targeting strategies to decrease the pathogenic potential of Colletotrichum species.
The enzyme is able to hydrolyze fatty acids esters and emulsified triacylglycerol as efficiently as lipases, and therefore, it is considered an intermediate between esterases and lipases (https ://www. ebi.ac.uk/enzym eport al/ec/3.1.1.74) (Nyyssölä, 2015). The enzyme plays an active role in (a) carbon acquisition for saprophytic growth, (b) adhesion of fungal structures to the host surface, and (c) the early stages of fungal penetration (Auyong et al., 2015). Cutin monomers trigger expression and synthesis of cutinases required for fungal penetration into the plant tissues. Cutinases, therefore, have a critical role in plant surface signalling that elicits differentiation of those fungal structures required for infection (Belbahri, Calmin, Mauch, & Andersson, 2008).
Bell pepper (Capsicum annuum var. grossum (L.) Sendt.) is a member of the Solanaceae family and is one of the most widely cultivated vegetable crops in the world. Over the last decade, the world production and consumption of bell peppers have been steadily increasing. More than 70% of the world's bell peppers are produced in China (FAO, 2017, http://www.fao.org/faost at/en/#data/QC). In Trinidad, bell pepper is grown year-round and is among the top 10 agricultural commodities in the country. Production, in terms of yield and cost, is directly affected by fruit rot caused primarily by C. truncatum (syn. C. capsici -Damm, Woudenberg, Cannon, & Crous, 2009;Ramdial & Rampersad, 2015) and more recently by C. brevisporum, albeit with lower incidence compared to C. truncatum (Villafana, Ramdass, & Rampersad, 2019). C. truncatum was the predominant species of Colletotrichum associated with anthracnose of chili in Asia and is widely distributed throughout Asia, Australia, and South America De Silva et al., 2019;Diao et al., 2017;Mongkolporn & Taylor, 2018;Sharma, Kumar Pinnaka, & Shenoy, 2014). Multilocus phylogeny revealed that the recently characterized Colletotrichum magnum species complex consists of nine closely-related species which includes C. brevisporum (Damm et al., 2019). C. brevisporum has also been reported as one of the causal agents of anthracnose disease in chili fruit in China and Brazil and in bell pepper fruit in Trinidad (De Silva, Crous, Ades, Hyde, & Taylor, 2017;Diao et al., 2017;Liu et al., 2016;Villafana et al., 2019). truncatum isolates as a major fungal pathogen of green and red bell pepper fruit worldwide. This work will increase our understanding of the evolution of cutinase genes in fungal-plant host interactions which can be used to develop strategies for disruption of cutinase gene target(s) to reduce diseases caused by these fungal pathogens. Sudan III was used to stain the cuticle an orange to red color for microscopic visualization (Yeung & Chan, 2015).

| Isolation of fungal pathogens
The bell pepper fruits were surface sterilized by rinsing in 70% ethanol for 1 min followed by another rinse in 0.6% sodium hypochlorite solution for 1 min. Samples were then washed three times in sterilized distilled water and dried on sterilized tissue paper. 4-mm 3 blocks of fruit tissue were removed from the margins of the lesions and transferred to potato dextrose agar (PDA) media (Oxoid Ltd., Thermo Fisher Scientific, Inc., USA) supplemented with 50 mg/L streptomycin, tetracycline, and chloramphenicol. Plates were incubated for seven days in the dark at 25°C. Monoconidial cultures of C. truncatum, and C. brevisporum were subsequently obtained and maintained on PDA at 4°C for temporary storage, and as conidial suspensions in 50% glycerol at −70°C for long-term storage. The identities of C. truncatum and C. brevisporum cultures were con-

| Tween-20 and tributyrin opacity test
Gel diffusion bioassays, for example, the Tween and tributyrin opacity test, are based on the ability of lipase-secreting microbes to break down the lipid substrate incorporated into solid media (see review by Lanka & Latha, 2015). These tests minimize the cost of screening and protect the test microorganisms from the inhibitory effects of various indicator dyes and lipase activity is identified as a clear or turbid zone around the colonies after incubation (Lanka & Latha, 2015). The level of enzyme activity can be evaluated by measuring the diameter of the halo around the colonies. In this study, for the Tween-20 test, a calcium salt-free Bacto agar (Oxoid Ltd., Thermo Fisher Scientific Inc.) medium containing Tween-20 (Sigma Aldrich Inc.) was used (Slifkin, 2000). A 4-mm 3 block taken from the advancing mycelial edge of an actively growing culture of each isolate (eight isolates each of C. truncatum, and C. brevisporum) was plated onto this medium in duplicate. The presence of a white crystalline precipitate around the colony indicated secreted lipase activity. The colony size was measured after three days. The test was repeated.

| Statistical analyses
Data were analyzed using IBM SPSS Statistics version 20. Tables outlining summary statistics for colony diameter (mm) on Tween-20 and tributyrin, respectively, as well as for halo size (mm) on tributyrin. Analysis of variance tests (One-way ANOVA) was carried out for "Colony diameter (mm)* Species" for Tween-20 and tributyrin samples, respectively, and "Halo size (mm)* Species" for tributyrin samples and the appropriate tables were extracted.

| Rhodamine test
Use of a fluorescent indicator dye such as Rhodamine B or G in the presence of olive oil as a lipid substrate has been used to determine lipase-positive microbes (Lanka & Latha, 2015). Hydrolysis of a lipid substrate (e.g., olive oil) by secreted lipase results in the production of free fatty acids which interacts with Rhodamine fluorescent dye in the medium and causes the formation of yellow to orange fluorescent colonies which are visible upon UV irradiation. A 4-mm 3 block taken from the advancing mycelial edge of an actively growing culture of each isolate (eight isolates each of C. truncatum, and C. brevisporum) was plated onto this medium in duplicate as previously described for the Tween-20 and tributyrin opacity tests. This test was repeated. Observation of a fluorescent yellow to orange colonies under UV light was recorded after six days.

| DNA extraction, PCR amplification, and sequencing
DNA was extracted from 21 actively growing colonies using the Maxwell ® -16 automated DNA extraction system (Promega) based on magnetic bead capture DNA extraction according to the manufacturer's instructions. PCR amplification was carried out using published protocols for culture identification with modifications (O'Donnell et al., 2004;White et al., 1990). The primers and their use in this study are described in Table 1. For a single 25 µl reaction using GoTaq® Green Master Hot Start Taq DNA Polymerase (Promega Corporation) the PCR components included 1 × master mix, 50 pmoles of each primer (Integrated DNA Technologies), and 5 µl of the 1:4 diluted DNA sample.
PCR amplification thermal conditions consisted of an initial denaturation of 5 min at 94°C followed by 35 cycles of 1 min at 94°C, annealing temperature and duration according to primer sequences, 1 min at 72°C with a final extension of 5 min at 72°C. Amplicons were sequenced at MCLAB (Molecular Cloning Laboratories). Sequence identities were verified using the BLAST algorithm in NCBI. Representative cutinase sequences for C. truncatum and C. brevisporum were deposited in GenBank (GenBank Accession Nos. MN473062 and MN473063, respectively). Twenty-one representative cccut-F/R-generated nucleotide sequences were used in the final data set.

| Cutinase amino acid sequence diversity
Based on the findings of the cluster analysis, nucleotide sequences of the cccut-F/R data set were translated to single amino acid sequence and the correct reading frame was identified using the ExPaSY translate tool (https ://web.expasy.org/trans late/). Two data subsets were analyzed: data subset 1-cutinase amino acid sequence (derived from translation of amplified nucleotide sequences by cccut-F/R primers) curated to 219 amino acids in length and which included exonic and intronic sequences; data subset 2-cutinase amino acid sequence (derived from translation of amplified nucleotide sequences by cccut-F/R primers) curated to 66 amino acids in length and only included exonic sequences. The alignment, query coverage, and percent identity were then examined for "within" Colletotrichum species (intraspecific diversity) and "among" Colletotrichum species (interspecific diversity).
Multiple nucleotide sequences were translated to amino acid sequences using the EMBOSS Transeq tool (https ://www.ebi.ac.uk/ Tools/ st/emboss_trans eq/). The multiple sequences were then aligned using EMBL-EBI Clustal Omega (https ://www.ebi.ac.uk/Tools/ msa/ clust alo/). Clustal Omega is a multiple sequence alignment program that uses seeded guide trees and HMM profile-profile techniques to generate alignments among three or more sequences. This alignment was then used to generate an amino acid conservation plot in BioEdit (Hall, 1999), and this conserved sequence was used in subsequent analyses. Protein domain superfamilies in CATH-Gene3D (http:// www.cathdb.info/searc h/; Dawson et al., 2016;Lewis et al., 2017) have been subclassified into functional families (or FunFams), which are groups of protein sequences and structures with a high probability of sharing the same function(s). Therefore, the functionally important residues in a family are also expected to be highly conserved.

| Lineage of cutinase protein based on sequence, structure, and functional diversity
CATH-Gene3D provides information on the evolutionary relationships of protein domains through sequence, structure, and  Grigoriev et al., 2011Grigoriev et al., , 2014.

| RE SULTS
Lesions caused by C. truncatum were recorded at the shoulder, face, and bottom of the fruit (Figure 1). The frequency of lesion according to location on fruit surface was 44.2% on the shoulder, 50.0% on the face and 53.8% on the bottom. As such, the frequency distribution showed no preference for lesion development at a specific location on the fruit surface (Table A1). Figure 2 shows the difference in cuticle integrity and thickness in green versus red bell pepper fruit.

| Cutinase bioassays
Tween-20 Opacity Test: A white crystalline precipitate was observed for all isolates of C. truncatum and C. brevisporum (Figure 3a). for all isolates plated on tributyrin agar indicated lipase activity for C.
truncatum and C. brevisporum. (Figure 3b). Summary of statistics for colony diameter (mm) for each Colletotrichum species on tributyrin media (Table A4) indicated that C. truncatum had the larger colony diameter (mm) at 15.56 mm, as well as the higher mean diameter value at 6.18 mm. The maximum and minimum halo sizes (mm) measured for C. truncatum were 12.74 mm and 3.42 mm, respectively. C. truncatum halo diameter also had higher mean value at 8.71 mm (Table A5).
Analysis of variance of colony diameter for the two Colletotrichum species indicated that there was a significant difference in colony diameter (mm) between C. truncatum and C. brevisporum, (p = .021; Table A6). However, there was no significant difference in halo diameter (mm) for C. truncatum and C. brevisporum (Table A7).
Rhodamine Test: Yellow-orange-colored fluorescent colonies were observed under UV light, which confirmed secreted cutinase activity for all isolates of C. truncatum and C. brevisporum (Figure 3c).

| Nucleotide sequence analysis
Nucleotide sequences generated from cccut-F/R primers (trimmed to a final length of 656 bases) were identified as cutinase gene with

| Cluster analysis
For the cccut-F/R-generated nucleotide sequence analysis, the clusters were strongly supported (minimum bs ≥ 85%) and were constructed according to haplotype: two haplotypes for the C. truncatum sequences, one haplotype for the C. brevisporum sequences, and two haplotypes that represented the two C. gloeosporioides sequences (Figure 4). The structure of each haplotype cluster was largely polytomic indicating no sequence diversity within a particular cluster. Clustering was irrespective of the maturity of the bell peppers fruit, that is, whether red or green.

| Cutinase amino acid sequence diversity
Cutinase amino acid sequence was invariable at the intraspecific level with 100% amino acid sequence identity at 100% coverage of the query sequence with C. truncatum (UniProtKB -P10951; GenBank Accession No. ADQ27862). However, variability existed at the interspecific level with 83.3% sequence identity to C. gloeosporioides (GenBank Accession Nos. AAL38030 and AKH80819) at 100% of the query sequence. "Among species" amino acid sequence variability was at minimum for C. gloeosporioides at 83.3% and maximum for C. spinosum at 74.2% (Table 3). For all C. truncatum and C. brevisporum amino acid sequences, there was 100% conservation of the core coding region that corresponded to the cutinase domain despite nucleotide sequence variation in the same region ( Figure 5).

| Conservation of cutinase based on 200 fungal amino acid sequences
An alignment of 200 truncated amino acid sequences of different fungal species with an identified cutinase domain in CATH-Gene3D protein data bank revealed specific regions in the sequence that may be indicative of "diversity hot spots" (Figure 6). This is in comparison to the conservation plot of aligned amino acid sequences of C. truncatum isolates from Trinidad and two reference C. truncatum sequences mined from GenBank which illustrated 100% conservation. The functionally important residues in this protein superfamily are expected to be highly conserved. CATH indicated that the name of this superfamily (3.40.50.1820), previously called alpha/ beta hydrolase, has been modified, and the official domain name is pending.

| Structure of cutinase gene
The structure of the cutinase gene and protein was analyzed across 34 available Colletotrichum genomes in the MycoCosm database. C. truncatum and C. brevisporum were not among the genomes available for reference in the MycoCosm repository. The predicted cutinase gene length for 34 Colletotrichum sequences in the MycoCosm database whose cutinase gene length ranged from 967 bp to 1,296 bp which included two exons interrupted by one intron. Among the 34 genomes, the cutinase gene was composed of two exons interrupted by one intron. Exon 1 varied from 343 to 733 bp in length, exon 2 varied from 556 to 786 bp in length, and the intronic sequence varied in length from 32 to 85 bp. ( Figure S1). There was, therefore, one splice variant where the intervening intron between the two coding regions is removed.

| Protein signatures
There was a highly conserved domain ( Figure S2), characteristic of serine hydrolase, consisting of 194 amino acid residues with a Ser, His, Asp catalytic triad, and 5-element fingerprint (motifs 1 to 5) among all Colletotrichum species. As such, the enzyme had three identified active sites, ACT_SITE140, ACT_SITE195, and ACT_SITE208 (https ://www.ebi.ac.uk/enzym eport al/searc h/P1095 1/enzyme), and whose source data are derived from IntEnz (Integrated relational   Figure S3).

| Organization and synteny of cutinase gene among Colletotrichum genomes
Synteny analysis in MycoCosm indicated that the cutinase domain was flanked by highly conserved amino acid sequences at both the N-and C-termini in the sequences of 34 Colletotrichum species (Figures 7-9). At the N-terminus, there was an uncharacterized but highly conserved hypothetical protein (KOG id 2306) whose amino acid sequence identity ranged from 69.3% ( At the C-terminus, a PPR domain (pentatricopeptide repeat) was identified whose function is hypothetical but predicted to involve RNA stabilization and processing as it retains an amino acid sequence-specific  Horton et al., 2007) was used in protein subcellular localization prediction which confirmed that the enzyme product, deduced from in-frame amino acid sequence of test isolates, is extracellular or secreted. The identified domains at the both termini were conserved both in their structure and strict arrangement immediate to and relative to the cutinase domain.

| D ISCUSS I ON
This study investigated the diversity, structure, organization, and synteny of a single cutinase gene within C. truncatum and C. brevisporum isolates from bell pepper fruit in Trinidad and among different F I G U R E 5 Conservation plot of cutinase amino acid sequence belonging to Colletotrichum truncatum isolates from Trinidad and C. truncatum reference sequences from GenBank | 1435

VILLAFANA ANd RAMPERSAd
Colletotrichum species. Evidence of secreted cutinase capability was first tested among the C. truncatum and C. brevisporum isolates from Trinidad based on bioassays using different lipid substrates. Rhodamine B was used as the determining factor for the presence of lipase activity (Lanka & Latha, 2015;Tomulescu et al., 2015). Bornscheuer (2002) explained that lipases can be differentiated from carboxyl esterases based on their substrate spectra: p-nitrophenyl palmitate (pNPP) is hydrolyzed by lipases; p-nitrophenyl butyrate (pNPB) is hydrolyzed by esterases. However, Chen, Liang, Zhang, and Rodrigues (2006) reported that C. kahawae and C. gloeosporioides isolates demonstrated cutinase activity on tributyrin media but this activity was significantly reduced with p-nitrophenyl butyrate assays. Conversely, Bonnen and Hammerschmidt (1989) stated that a purified cutinase enzyme, extracted from C. lagenarium isolates, was active on both cutin and p-nitrophenylbutyrate as substrates. For this reason, three bioassays using different lipid substrates were used to assess cutinase activity in this study.

| Cutinase nucleotide and amino acid sequence variation among only C. truncatum
Cluster analysis indicated a low level of nucleotide variation among C. truncatum sequences. Wang et al., (2017) also found high homology among cutinase (CUT1) gene sequences of C. gloeosporioides.
The cutinase nucleotide sequences of C. brevisporum in this study, were more related to C. truncatum cutinase nucleotide sequences than to C. gloeosporioides. Cluster patterns coincided with haplotype with moderate to high bootstrap support. Zhu and Freeland (2006) purported that the degenerate nature of the genetic code enables amino acid changes that conserve certain amino acid properties. As such, the 66-amino acid sequence translated from the partial coding region of the cutinase gene was highly conserved within C. truncatum and C. brevisporum species. Pathogenesis is actualized through cutinase production and secretion among C. truncatum isolates, therefore, evolutionary maintenance of those amino acids in the primary protein structure that are important for folding, structural stability, and are required to form a substrate binding site(s) which guarantees the catalytic ability of the enzyme (Altenhoff et al., 2019;Rudnicki, Mroczek, & Cudek, 2014).

| Cutinase sequence diversity
The cutinase gene family in various genera of fungal genomes demonstrates a high degree of coding-sequence variation reflective of the diverse of roles attributed to cutinases and which perhaps, allowed adaptation to diverse ecological niches over time (Deng, Carbone, & Dean, 2007;Skamnioti et al., 2008). However, the relative degree of nucleotide and amino acid sequence diversity in addition to the role of these genes and their gene products in pathogenesis can vary (Liu et al., 2016;Skamnioti & Gurr, 2007;Sweigard, Chumley, & Valent, 1992). For example, in M. oryzae, CUT1 gene is essential for pathogenicity and CUT2 gene is required for cuticle sensing and formation of infection structures (Skamnioti et al., 2008). It is also reasonable to associ-

| Orthologue detection in 200 fungal genomes and implications of gene duplications
Twenty cutinase orthologues detected among different fungal species and whose common ancestor is Pezizomycotina were identified in this study. Using the CATH-Gene3D hierarchical classification approach which grouped cutinase protein domains according to sequence, structure, and functional diversity, confident predictions of the likely evolutionary relatives indicated that a hypothesized gene duplication event is one mechanism by which the cutinase gene in C. truncatum evolved. Gene duplication is an evolutionary tool by which new genes and genetic novelty can be generated in eukaryotes which in turn, would serve to increase the plasticity of a genome or species as an adaptive advantage to changing environments. Several factors impact upon whether a duplicated copy of this gene is fixed in the lineage of given pathogen population through positive selection forces. Primarily, the copy must serve some selective advantage to the pathogen, for example, two copies of one gene may increase the rate of expression and production of gene products or the duplicated copy may have evolved through neofunctionalization and is now adjunct to the function of the original gene (Magadum, Banerjee, Murugan, Gangapur, & Ravikesavan, 2013;Savory, Leonard, & Richards, 2015). Among test isolates in this study, the aligned cutinase nucleotide sequences, generated from the cccut-F/R primer pair, demonstrated signatures of significant positive selection according to Fu and Li's D* statistic which indicates the importance of this gene's functions during host pathogen interaction (Tiffin & Moeller, 2006).
It is suggested that the hypothesized lineage of the cutinase gene emerged after its transference between distantly related plant-degrading microbes through lateral gene transfer events (LGTs) (Belbahri et al., 2008). Genes that evolved from what is considered to be an "ancient" duplication event (i.e., duplication before speciation as in the case of Pzezizomycotina and subsequent spe-

| Synteny among 34 Colletotrichum genomes
It is useful to investigate synteny, in the context of genome structure evolution, because it demonstrates the comparative framework by which conservation of protein-coding genes and gene order are enabled between genomes of different species. This type of analysis is carried out under the presumption that genome assembly of good contiguation is used in the analysis (Liu, Hunt, & Tsai, 2018). The findings revealed that the cutinase domain was retained both in structure and arrangement among Colletotrichum species. The order of aligned genomic blocks between species and the arrangement of flanking protein domains were also conserved and shared for those domains immediately located at the N-and C-terminus of the cutinase domain. No synteny is suggestive of genomic rearrangements during speciation in order to colonize different niches (Bhadauria, Vijayan, Wei, & Banniza, 2017). Among the conserved domains that flanked the cutinase block were signal peptide and chaperone proteins. One explanation for this finding is the production of cutinase is enhanced by signal peptide optimization and chaperone expression (Yao, Su, Li, & Wu, 2019). It is important that the function of the highly conserved region located at the immediate N-terminus of the cutinase domain found in this study, be characterized because its high interspecific conservation in arrangement strongly suggests a contributory role in cutinase expression and function among Colletotrichum species. Further, these conserved genomic blocks can be alternative targets for cutinase function disruption and possibly disease control.
In this study, it was found that positive selection of low diversity cutinase gene sequence, preservation of amino acid sequence with conservation of protein function and synteny have been identified as evolutionary mechanisms that maintain the cutinase gene in the genomes of Colletotrichum species. This information is important to developing strategies to enhance plant defense and decrease the pathogenic potential of fungal phytopathogens by targeting and disrupting the cutinase genomic block identified in this study for Colletotrichum species (Brunner et al., 2013). This may be particularly important for the management of C. truncatum diseases as there is only one cutinase gene in C. truncatum available for targeted gene disruption.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.