Multi-Locus Phylogenetic Analysis Revealed the Association of Six Colletotrichum Species with Anthracnose Disease of Coffee (Coffea arabica L.) in Saudi Arabia

Several Colletotrichum species are able to cause anthracnose disease in coffee (Coffea arabica L.) and occur in all coffee production areas worldwide. A planned investigation of coffee plantations was carried out in Southwest Saudi Arabia in October, November, and December 2022. Various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. Isolation from symptomatic fresh samples was performed on a PDA medium supplemented with streptomycin sulfate (300 mg L−1) and copper hydroxide (42.5 mg L−1). Twenty-seven pure isolates of Colletotrichum-like fungi were obtained using a spore suspension method. The taxonomic placements of Colletotrichum-like fungi were performed based on the sequence dataset of multi-loci of internal transcribed spacer region rDNA (ITS), chitin synthase I (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB2), and partial mating type (Mat1–2) (ApMat) genes. The novel species are described in detail, including comprehensive morphological characteristics and colored illustrations. The pathogenicity of the isolated Colletotrichum species was assessed on detached coffee leaves as well as green and red fruit under laboratory conditions. The multi-locus phylogenetic analyses of the six-loci, ITS, ACT, CHS-1, TUB2, GAPDH and ApMat, revealed that 25 isolates were allocated within the C. gloeosporioides complex, while the remaining two isolates were assigned to the C. boninense complex. Six species were recognized, four of them, C. aeschynomenes, C. siamense, C. phyllanthi, and C. karstii, had been previously described. Based on molecular analyses and morphological examination comparisons, C. saudianum and C. coffeae-arabicae represent novel members within the C. gloeosporioides complex. Pathogenicity investigation confirmed that the Colletotrichum species could induce disease in coffee leaves as well as green and red fruits with variations. Based on the available literature and research, this is the first documentation for C. aeschynomenes, C. siamense, C. karstii, C. phyllanthi, C. saudianum, and C. coffeae-arabicae to cause anthracnose on coffee in Saudi Arabia.


Introduction
The genus Coffea is a member of the family Rubiaceae and is indigenous to the African continent, specifically Ethiopia [1]. Under this genus, there are two subgenera, Coffea and Baracoffea, which together comprise about 103 species [2]. Among all the species, the two most common and economically grown commercial species worldwide are C. canephora (Robusta) and C. arabica L. (Arabica). Historically, the coffee species could be traced to the Kaffa region of Ethiopia, and were later introduced to other parts of the world by traders

Sampling and Isolation
Coffee plantations were surveyed during October, November, and December 2022 in Jazan, Al Baha, Najran, and Asir regions (Table 1). Eighty-five vegetative samples from various tree parts, including fruits, leaves, and twigs, showing anthracnose symptoms were collected. Isolation from plant samples was made after surface disinfection through successive washing in 70 % ethanol for 30 s, followed by a 1 min wash in household bleach containing 1% NaOCl, and finally rinsed in distilled sterilized water and were dried using sterile filter paper [20]. Small pieces measuring 2-5 mm 2 , located between the infected and healthy tissues, were placed on potato dextrose agar medium (PDA) supplemented with streptomycin sulfate (300 mg/L −1 ) and copper hydroxide (42.5 mg/L −1 ) to inhibit bacterial and some fungal contamination [32]. Under dark conditions, the plates were incubated at 25 • C until the growth of fungi became visible. To obtain purified cultures, a hyphal tip was excised from the margins of the colonies that had developed from the tissue fragments and placed onto a new PDA medium. The new PDA medium was then incubated under the same conditions. Subsequently, single spore isolates were obtained using a spore suspension method [33]. The total genomic DNA was obtained from the harvested fresh mycelium of 7-day old cultures of Colletotrichum-like isolates grown on a PDA medium using the Dellaporta protocol for genomic DNA isolation [34]. Six gene regions, comprising the 5.8S nuclear ribosomal gene with two flanking internal transcribed spacers (ITS), chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as partial mating type (Mat1-2) (ApMat) genes were amplified and sequenced. These gene regions were amplified with the primer pairs ITS1 + ITS4 for ITS [35], ACT-783R + ACT-512F for ACT act [36], T1 [37] + Bt2b [38] for TUB2, GDF + GDR for GAPDH [39], and AMF1 and AMR1 for ApMat [29], respectively. The primers that were utilized to amplify and sequence the DNA of Colletotrichum isolates in this study are shown in Table 2. The PCR reaction was carried out in 25 µL reaction volume, comprising 10 µL PCR Master Mix (amaR OnePCR, GeneDirex, Inc., Las Vegas, NV, USA), 1 µL of template DNA, 1.5 µL from each primer, and 11 µL of ddH 2 O. The PCR was carried out using a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), and the amplification conditions for ITS, CHS-1, ACT, GAPDH, and TUB2 were identical to those outlined by Damm et al. [27]. For the ApMat gene, we followed the PCR amplification conditions outlined by Silva et al. [29]. The generated PCR products underwent bidirectional sequencing via Macrogen (Seoul, Republic of Korea) in accordance with the manufacturer's guidelines.

Phylogenetic Analyses
All obtained sequences underwent nucleotide BLAST search engine via the NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 22 February 2022)) to check the potential similarity with the closely related taxa. The new released sequences were aligned with the nucleotide sequences of reference strains of Colletotrichum (Table 3) belonging to the same complex retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov (accessed on 28 February 2022)), based on recent publications [23,[40][41][42]. The taxonomic identity of the strains was investigated by phylogenetic analysis of combined gene regions. For the C. boninense species complex, the ACT, ITS, TUB2, and CHS-1 were utilized, while ITS, ACT, CHS-1, TUB2, GAPDH, and ApMat combined gene regions were employed for the C. gloesporioides species complex. MEGA XI v.11.0.8 was utilized for trimming and concatenating the multi-sequence alignment. The C. gloesporioides complex alignment has 113 taxa with 2905 characters, 681 parsimony-informative, 1535 distinct patterns, 527 constant sites, and 1697 singleton sites. The C. boninense complex alignment has 36 taxa with 1448 characters, 478 distinct patterns, 224 parsimony-informative, 214 singleton sites, and 1010 constant sites. IQ-TREE multicore version 2.2.0 [43] was employed to calculate the best-fit evolution model based on BIC by ModelFinder [44] and to infer the phylogenetic tree Maximum likelihood (ML) relying upon 10,000 ultrafast bootstrap support replicates [45] on the partitioned dataset [46].       The combined partitioned dataset with adapted substitution models was subjected to Bayesian analysis using MrBayes v3.2.6 on Cipres Science Gateway (www.phylo.org) (accessed on 22 February 2022), adapted by the previously ModelFinder calculation. The analysis was conducted in duplicate using four Markov chain Monte Carlo (MCMC) chains for 10,000,000 generations, and random trees sampling for every 1000 generations. During the Bayesian analysis, a temperature value of 0.10 and a burn-in of 0.25 were used. The analysis was set to stop automatically once the split frequencies' average standard deviation became less than 0.01. For the C. boninense complex, we used 1210 samples from two runs, each of which yielded 806 samples, from which 605 were selected for the final analysis. For the C. gloesporioides complex, we used 6894 samples from two runs, each of which yielded 4596 trees, from which 3447 were sampled. The ML and Bayesian phylogenetic trees were viewed in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree (accessed on 15 March 2022)).

Morphological Characterization
Morphological characterization of Colletotrichum species was carried out as previously published [8,27]. For each characteristic isolate, the shape and sizes of 50 conidia were documented. In addition, the conidiophores, seta, and appressoria measurements were made for at least 30 at 100×magnification using Leica DM2500 LED light microscope with interference contrast (DIC). Appressoria was produced by dropping approximately 50 µL of conidial suspension on a glass slide, fixing the cover slip, and incubating for 5 days at 25 • C within a moist chamber. The results are presented as the minimum and maximum values along with the mean value ± its corresponding standard deviation (SD) for all measurements. Description and illustrations of novel species of Colletotrichum were deposited in MycoBank [47].

Pathogenicity Tests
Koch's postulates were applied, and pathogenicity was carried out under controlled laboratory conditions on detached leaves and fruits of Coffea arabica [20]. Selected isolates representing six Colletotrichum species were first grown for 7 days on a PDA medium at 25 • C. Leaves and fruits that were of equal size and age and in good health were chosen for the inoculation process. Leaves and fruits were subjected to surface disinfection with household bleach (NaOCl 1%) for a 2 min period before washing in sterile distilled water and air-drying. To ensure the accuracy of the experiment, six replicates were carried out for each isolate. Each replicate involved three leaves and five fruits. The leaves were gently punctured at three points on the midrib's upper surface utilizing a sterile needle tip. Coffee fruits were wounded by pinpricking the fruit wall to approximately 1 mm depth. Using the actively growing margins of each isolate, 5 mm of mycelium plug was extracted and positioned onto the wounded sites. The control leaves were subjected to inoculation using solely sterile PDA plugs. After inoculation, the leaves and fruits were then transferred into plastic boxes with lining of wetted paper towels to maintain high relative humidity. These were then incubated for 5-7 days at 25 • C, while being observed every day to detect the development of any symptoms. This experiment was repeated twice.

Data Analysis
Statistical analysis of variance [48] was achieved through employing SPSS 16.0 statistical package (SPSS Inc., Chicago, IL, USA) to delineate the mean size ± SD (standard deviation) of lesion diameters. Discrepancies in lesions diameters were documented after performing one-way-ANOVA at p < 0.05 and 95% confidence level. The mean of the measured values was compared utilizing the Least Significant Difference (LSD) test (p < 0.05).

Symptoms Observation and Isolation
The coffee trees' young leaves exhibited visible symptoms of anthracnose in the form of randomly scattered minor, irregular brown to black lesions. These lesions could expand and merge, leading to the formation of necrotic black patches ( Figure 1A,B), which gave leaves a scorched appearance. The necrotic tissues were usually cracked forming holes on the leaf blade and finally detached from branches. On the twigs, black speaks initially starting from the apical portion and extended along the twig surface, leading to the death of the apical and lateral shoots ( Figure 1C). Upon observing the semi-immersed fruiting structures (acervuli), orange masses of conidia were detected on the necrotic tissues that were released. Prominent, sunken dark decay lesions could extend deeply into the fruit, ultimately leading to the decay of fruit pulp of green and red berries ( Figure 1D-F). In total, 27 Colletotrichum-like isolates were obtained; 18 from leaves, 6 from fruit, and 3 from branches (Table S1). The phylogenetic study comprised all the isolates obtained.

Molecular Characterization
The identification of all Colletotrichum-like isolates began with their classification up to the genus level, which relies upon their ITS sequences. Identity of isolates was further confirmed at the species level, based on the multi-locus phylogenetic analysis of the sixloci (ITS, ACT, CHS-1, TUB2, ApMat, and GAPDH) for our 27 sequences of Colletotrichum isolates along with reference sequences retrieved from GenBank (Table 3). This analysis revealed that 27 isolates were assigned into two species complexes, the C. gloeosporioides complex and C. boninense complex. Among the 27 isolates, 25 allocated within the C. gloeosporioides complex, and the remaining two belonged to the C. boninense complex. In the phylogenetic tree (Figure 2) of the six-loci ITS, ACT, CHS-1, TUB2, ApMat, and GAPDH, 25 isolates within the C. gloeosporioides complex clustered in four clades, eight of them with C. siamense and single isolate with C. aeschynomenes. Furthermore, two discrete clades were positioned far apart from all recognized species within the complex, and thus, they were recognized as new species and named C. saudianum and C. coffeae-arabicae (Figure 2). In the C. boninense complex phylogenetic tree (Figure 3), each of the two isolates were grouped in distinct clade. The phylogenetic analysis strongly supported the placement of PPDU41K in a clade with CBS129833, VPRI43652, and CBS126532 of C. karstii, as indicated by the high BS/BPP values (100%/1.0). This clade was recognized as C. karstii on the phy-

Molecular Characterization
The identification of all Colletotrichum-like isolates began with their classification up to the genus level, which relies upon their ITS sequences. Identity of isolates was further confirmed at the species level, based on the multi-locus phylogenetic analysis of the six-loci (ITS, ACT, CHS-1, TUB2, ApMat, and GAPDH) for our 27 sequences of Colletotrichum isolates along with reference sequences retrieved from GenBank (Table 3). This analysis revealed that 27 isolates were assigned into two species complexes, the C. gloeosporioides complex and C. boninense complex. Among the 27 isolates, 25 allocated within the C. gloeosporioides complex, and the remaining two belonged to the C. boninense complex. In the phylogenetic tree (Figure 2) of the six-loci ITS, ACT, CHS-1, TUB2, ApMat, and GAPDH, 25 isolates within the C. gloeosporioides complex clustered in four clades, eight of them with C. siamense and single isolate with C. aeschynomenes. Furthermore, two discrete clades were positioned far apart from all recognized species within the complex, and thus, they were recognized as new species and named C. saudianum and C. coffeae-arabicae (Figure 2). In the C. boninense complex phylogenetic tree (Figure 3), each of the two isolates were grouped in distinct clade. The phylogenetic analysis strongly supported the placement of PPDU41K in a clade with CBS129833, VPRI43652, and CBS126532 of C. karstii, as indicated by the high BS/BPP values (100%/1.0). This clade was recognized as C. karstii on the phylogenetic tree ( Figure 3). The second isolate, PPDU36S, was grouped with the isolate CBS175.67 of C. phyllanthi within a clade highly supported with BS/BPP values (90%/1.0). Therefore, PPDU36S was identified as the known species C. phyllanthi.

Taxonomy
The morphological characteristics and multi-locus phylogeny helped designate th 27 isolates attained in this study into six distinct species. Four species, C. aeschynomene C. siamense, C. karstii, and C. phyllanthi, were firstly documented from coffee in Saudi Ar bia, and a further two species were newly described.

Taxonomy
The morphological characteristics and multi-locus phylogeny helped designate the 27 isolates attained in this study into six distinct species. Four species, C. aeschynomenes, C. siamense, C. karstii, and C. phyllanthi, were firstly documented from coffee in Saudi Arabia, and a further two species were newly described.
Culture characteristics: the colonies on PDA are fluffy with white raised cottony mycelia, turned dark mouse-grey in the center, pale grey with an entire margin. The reverse of the colonies is iron grey to olivaceous grey. Following a 7-day incubation at 25 °C in the dark, the colonies grown to the Petri plate edge, measuring 85 mm. The conidia appear as pinkish-orange masses released from semi-immersed acervuli.
Culture characteristics: the colonies grown on PDA were sparse and dense, with effuse mycelium mats that were initially white and became olivaceous buff to greenish olivaceous on the upper surface. On the reverse side, the colonies had iron grey to olivaceous grey color. The color darkened with age. Following 10 days of dark incubation at 25 • C, the colonies grown to the Petri plate edge, measuring 85 mm. Conidia were observed as orange masses released from semi-immersed acervuli.
Etymology: The name refers to the host plant (Coffea Arabica) from where the fungus was originally collected.
Culture characteristics: the colonies on PDA are fluffy with white raised cottony mycelia, turned dark mouse-grey in the center, pale grey with an entire margin. The reverse of the colonies is iron grey to olivaceous grey. Following a 7-day incubation at 25 • C in the dark, the colonies grown to the Petri plate edge, measuring 85 mm. The conidia appear as pinkish-orange masses released from semi-immersed acervuli.

Pathogenicity Tests
Pathogenicity test results demonstrated that all the tested Colletotrichum isolates were able to induce disease symptoms similar to that recognized in the field on coffee leaves and fruits (Figures 6 and 7). After 5 days, small brown lesions appeared nearby the inoculation site, which then grew and developed into large necrotic brown lesions with black margins (Figure 7A-D). Orange conidial masses have been recognized on the surface of necrotic lesions on leaves as well as on red fruit after 12 days ( Figure 7D,F). No symptoms developed on the control leaves and fruits. The tested isolates of C. saudianum and C. siamense developed lesions 3 days earlier than the two isolates of C. karstii and C. phyllanthi, which developed lesions after 8 days. The LSD test revealed significant (p < 0.05) Notes: The C. gloeosporioides species complex is characterized by cylindrical conidia that have rounded ends and taper slightly towards the base, which is similar to the conidial morphology observed in C. coffeae-arabicae [8,25]. However, the multi-locus phylogenetic analysis revealed that the four C. coffeae-arabicae strains formed a discrete clade and were phylogenetically distinct from the current recognized species within the gloeosporioides complex. Furthermore, BLASTn search of the ex-type strain PPDU26B of C. coffeae-arabicae sequences revealed a variable sequence resemblance with other sequences within the NCBI GenBank from different species. The closest matches using the ITS had a 100% similarity to C. siamense (GenBank MT450691, MT450690, and MT450689). Furthermore, the closest ACT sequence match showed 100% similarity to C. aenigma (GenBank OQ698783 and OQ698782) and 100% to C. siamense (OQ698755). However, TUB2 showed the highest similarity 100% to C. siamense (GenBank OP660847; OP660836 and OP660829). However, the CHS-1 sequence revealed homology of 99.5% to C. gloeosporioides (GenBank MF554932 and ON723793) and 99% to C. fructicola (GenBank OQ703570). Moreover, the GAPDH sequences demonstrated 100% to C. siamense (GenBank MF110865; MN228537 and MN228536). Additionally, the ApMat sequences had 96.7% similarity with C. siamense (GenBank KX578771), 96.3 % with C. siamense (GenBank MW557490), and 96.1% with C. siamense (GenBank OM816816). The morphological comparisons and phylogenetic analyses ascribed C. coffeae-arabicae as a novel taxon within the C. gloeosporioides complex.

Pathogenicity Tests
Pathogenicity test results demonstrated that all the tested Colletotrichum isolates were able to induce disease symptoms similar to that recognized in the field on coffee leaves and fruits (Figures 6 and 7). After 5 days, small brown lesions appeared nearby the inoculation site, which then grew and developed into large necrotic brown lesions with black margins (Figure 7A-D). Orange conidial masses have been recognized on the surface of necrotic lesions on leaves as well as on red fruit after 12 days ( Figure 7D,F). No symptoms developed on the control leaves and fruits. The tested isolates of C. saudianum and C. siamense developed lesions 3 days earlier than the two isolates of C. karstii and C. phyllanthi, which developed lesions after 8 days. The LSD test revealed significant (p < 0.05) differences in lesion diameter induced by the tested isolates, of which C. saudianum PPDU38H caused the largest lesion diameter (1.63 cm), followed by C. saudianum PPDU28E, which produced lesion that reached 1.48 cm. Conversely, the remaining Colletotrichum isolates produced lesions that insignificantly (p < 0.05) varied in size from each other ( Figure 6A). The majority of isolates produced larger lesion sizes on red fruit than green ones ( Figure 7E, F), with the largest lesions caused by C. siamense PPDU27M (1.8 cm), C. saudianum PPDU38H (1.68 cm), C. saudianum PPDU28E (1.5 cm), and C. coffeae-arabicae PPDU29F (1.48 cm). In contrast, the smallest lesion sizes were caused by isolates C. aeschynomenes PPDU28A (0.88 cm), C. siamense PPDU40G (0.8 cm), C. karstii PPDU41K (0.5 mm), and C. phyllanthi PPDU36S (0.4 cm). On the other hand, the two isolates C. coffeae-arabicae PPDU29F and C. saudianum PPDU38H showed equal virulence on green fruit by producing similar lesion lengths (0.93, 0.9 cm, respectively), which were significantly (p < 0.05) larger than those of other isolates ( Figure 6B). Contrariwise, both C. karstii PPDU41K and C. phyllanthi PPDU36S revealed much lowered lesion expansion rate around the inoculation site over the experimental progress either on leaves or green as well as red fruits ( Figures 6A,B and 7). The differences in lesion diameters among Colletotrichum species and even isolates of the same species attributed to their geographical origin or the plat part where they were isolated. It was also observed that mature fruits were more sensitive than green ones and exhibited larger lesions diameters. The artificial inoculation of Colletotrichum species onto detached coffee leaves and fruits resulted in the successful recovery of the fungi, fulfilling Koch's postulates.
J. Fungi 2023, 9, x FOR PEER REVIEW 17 of 23 differences in lesion diameter induced by the tested isolates, of which C. saudianum PPDU38H caused the largest lesion diameter (1.63 cm), followed by C. saudianum PPDU28E, which produced lesion that reached 1.48 cm. Conversely, the remaining Colletotrichum isolates produced lesions that insignificantly (p < 0.05) varied in size from each other ( Figure 6A). The majority of isolates produced larger lesion sizes on red fruit than green ones ( Figure  On the other hand, the two isolates C. coffeae-arabicae PPDU29F and C. saudianum PPDU38H showed equal virulence on green fruit by producing similar lesion lengths (0.93, 0.9 cm, respectively), which were significantly (p < 0.05) larger than those of other isolates ( Figure 6B). Contrariwise, both C. karstii PPDU41K and C. phyllanthi PPDU36S revealed much lowered lesion expansion rate around the inoculation site over the experimental progress either on leaves or green as well as red fruits ( Figures 6A,B and 7). The differences in lesion diameters among Colletotrichum species and even isolates of the same species attributed to their geographical origin or the plat part where they were isolated. It was also observed that mature fruits were more sensitive than green ones and exhibited larger lesions diameters. The artificial inoculation of Colletotrichum species onto detached coffee leaves and fruits resulted in the successful recovery of the fungi, fulfilling Koch's postulates.

Discussion
Colletotrichum is a genus that comprises economically significant pathogenic species with numerous host plants worldwide. Few efforts have been made to assess the disease problems of Coffea arabica in Saudi Arabia. Therefore, this study represents the initial attempt to evaluate the occurrence and the diversity of Colletotrichum species that are linked to different symptom patterns recognized in coffee trees. During a planned survey carried out in October, November, and December 2022, various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. The wellknown anthracnose symptoms were often observed on the leaves as minute black to dark brown lesions with asymmetrical margins. Infections on the twigs and branches typically start from the apical portion along the twig surface, leading to the death of the apical and lateral shoots. Green and red berries exhibited dark, sunken, prominent lesions that deeply extended into the fruit, causing the fruit pulp to decay. These observed symptoms coincided with those previously reported [19,49].

Discussion
Colletotrichum is a genus that comprises economically significant pathogenic species with numerous host plants worldwide. Few efforts have been made to assess the disease problems of Coffea arabica in Saudi Arabia. Therefore, this study represents the initial attempt to evaluate the occurrence and the diversity of Colletotrichum species that are linked to different symptom patterns recognized in coffee trees. During a planned survey carried out in October, November, and December 2022, various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. The wellknown anthracnose symptoms were often observed on the leaves as minute black to dark brown lesions with asymmetrical margins. Infections on the twigs and branches typically start from the apical portion along the twig surface, leading to the death of the apical and lateral shoots. Green and red berries exhibited dark, sunken, prominent lesions that deeply extended into the fruit, causing the fruit pulp to decay. These observed symptoms coincided with those previously reported [19,49].
Accurate delineation of the causal organisms responsible for Colletotrichum infections is crucial, given the significant economic losses experienced by coffee plantations and the restricted knowledge of growers in this regard. In the present study, the ITS sequence data aided in placing the 27 isolates in the C. gloeosporioides and C. boninense species complexes, approving the usefulness of ITS sequencing for categorizing Colletotrichum isolates [24,50]. Furthermore, extensive phylogenetic inference depending upon multi-locus analyses of ITS, ACT, TUB2, CHS-1 GAPDH, and ApMat provided a firm resolution and allocated all Colletotrichum isolates associated with Coffea arabica into two distinct species complexes and additionally ascribed them into six species. Among the six species identified, four were already known, C. siamense, C. aeschynomenes, C. karstii, and C. phyllanthi, while two novel species, C. saudianum and C. coffeae-arabicae, were also identified. It was not easy to discriminate species of C. gloeosporioides complex depending upon the data of the five loci including, ITS, ACT, CHS-1, TUB2, and GAPDH. Interestingly, relying on the sequence data of the single gene ApMat adequately provided a robust separation between the species of the C. gloeosporioides complex, and the resulting tree has topology resembling the tree obtained by the six loci. It also aided in the confirmation of the identity of two newly described species in this study, namely C. saudianum and C. coffeae-arabicae. Our results are supported by those published by de Silva et al. [29], who confirmed that the ApMat marker solely was ultimately useful in disentangling species of the C. gloeosporioides complex isolated from C. arabica and other coffee species. Other studies have confirmed these findings. For example, Liu et al. [41] verified that the ApMat marker, along with GS, offers significant phylogenetic information and successfully separated 22 species in the C. gloeosporioides complex when compared to other used loci ITS, ACT, CHS-1, TUB2, GS, and GAPDH. In addition, the research of Khodadadi et al. [24] revealed that the ApMat, when combined with ITS and TUB2, could efficiently allocate the new species C. noveboracense to a discrete clade that was highly supported with Bayesian posterior probability and bootstrap values. Crouch et al. [51] first introduced the Apn2-Mat1 locus for differentiating species in the C. graminicola complex. This ApMat marker was subsequently used to separate species in the C. gloeosporioides complex [28,[52][53][54]. Both GAPDH and TUB2 markers are widely considered highly effective barcodes for most Colletotrichum complexes and are widely used. However, complex-specific barcodes must still be utilized in conjunction with them to achieve accurate species delimitation [8,28,29]. In our case study, GAPDH and TUB2 sequence did not consistently delineate species within the cryptic species of gloeosporioides complex. Accordingly, using ApMat sequence data approved the affordability and reliability of this marker for differentiating species of C. gloeosporioides complex. Therefore, we recommend combining ApMat with other markers as a sufficient technique for classifying species within the C. gloeosporioides complex.
Based on the results of this study, the most frequently reported species belonging to the C. gloeosporioides complex were C. siamense, C. aeschynomenes, C. saudianum, and C. coffeae-arabicae. Only two isolates representing two species, C. karstii and C. phyllanthi, belonged to the C. boninense species complex, and these were separated at much lowered frequency (one isolate for each). Among the species of C. gloeosporioides isolated from coffee, Colletotrichum saudianum (12 isolates) was the most frequently isolated, followed by C. siamense (8 isolates) and C. coffeae-arabicae (4 isolates). In contrast, only a single isolate of C. aeschynomenes was recovered. The presence of six species of Colletotrichum associated with anthracnose disease on coffee indicates that more than one Colletotrichum species can colonize a single host, which is consistent with the conclusion of previous studies [16,19,25,27,55]. The compositions of Colletotrichum species from coffee appeared to differ according to the geographical origin, host, and species complex. For example, C. kahawae also appears to be host-specific to Coffea species and geographically restricted and widespread in the African continent or in low altitudes [8,11,15]. However, C. kahawae has been reported to cause anthracnose disease on different hosts in Australia, Europe, South Africa, and USA [8,56]. Furthermore, other members of the C. gloeosporioides complex, such as C. siamense and C. fructicola, are widely reported in coffee in several countries and are known to have a broader host range. Although several species have been reported to cause infection in coffee, the association of C. aeschynomenes and C. phyllanthi and the newly described species C. saudianum and C. coffeae-arabicae is considered the first report in Saudi Arabia and worldwide. The low incidence of C. karstii and C. phyllanthi and the fact that the only two isolates of these species induced the smallest lesions on coffee leaves and fruit indicate that these species are of little importance and do not contribute significantly to anthracnose disease. Previous studies have reported that Colletotrichum karstii is a causal agent of anthracnose disease on coffee in Vietnam and Mexico, but in low frequencies [18][19][20], which supports our results. Colletotrichum phyllanthi, on the other hand, has not been previously reported on coffee, and we report for the first time its association with anthracnose symptoms.
Koch's postulates were fulfilled, indicating that all isolates were pathogenic to detached coffee leaves as well as green and red fruit with significant p < 0.05 variations in infection degree. Variations were also among isolates of the same species, with the most virulent species being C. saudianum, C. siamense, and C. coffeae-arabicae, which frequently recovered from coffee. On the other hand, the lowest dominant species, C. aeschynomenes, C. karstii, and C. phyllanthi, provoked the smallest lesions either on detached leaves or on fruit ( Figure 6). According to the statistical analysis, there were significant differences between isolates. These differences could be attributed to the geographical origin of isolates or/and plant part where it was isolated. The leaf lesions caused by the six Colletotrichum species were similar; however, the symptoms development and lesion sizes varied among species. For example, leaves and fruit inoculated with C. saudianum and C. siamense developed lesions 5 days earlier and larger than the other species, whereas the two isolates of C. karstii and C. phyllanthi developed lesions after 8 days. Similar results were also reported, in which the C. siamense was faster in developing lesions on coffee leaves and C. karstii was the slowest species, which produced lesions after 30 days of inoculation [19]. Additionally, Cao et al. [20] found out that among tested Colletotrichum species; C. siamense, C. gigasporum and C. karstii were the most virulent on both Arabica and Robusta coffee red fruits and recorded the same infection incidence 100 %. While on green fruit, the infection incidence was lower and registered 50, 0, and 25 %, respectively. Moreover, Nguyen et al., [57], indicated that C. fructicola and C. siamense can induce lesions on detached green berries after inoculation; however, the efficacious infection rate was low. In a similar study, Prihastuti et al. [16] demonstrated that C. fructicola was the most virulent species in producing higher infection percentage (89.93 %) on red fruit than C. asianum (63.06%) and C. siamense (50.19%). Similarly, Waller et al., [11] indicated that C. gloeosporioides isolates from coffee are capable of causing disease only on ripe berries, leaves, and are not able to cause the infection of green berries. These findings were also confirmed in laboratory trials in Papua New Guinea, of which C. gloeosporioides only infected ripe red berries [58]. These results supported our findings, of which the red fruits were more severely affected than green ones. The reasons behind this could be the onset of senescence, which are characterized by reduced defensive systems, weakened tissues, and increased ethylene production.

Conclusions
Understanding the taxonomy and the pathogenicity of Colletotrichum is fundamental in coffee production regions in order to manage this economically important disease and secure the profitability of the coffee industry in Saudi Arabia. Knowing the distribution of Colletotrichum species could help to propose a suitable control program based on their sensitivity to fungicides. In this study, ITS, TUB2, ACT, CHS-1 were sufficient to distinguish C. karstii and C. phyllanthi within the C. boninense complexes. In contrast, ITS, TUB2, ACT, CHS-1, GADPH, and ApMat regions were fundamental to differentiate species within the C. gloeosporioides complex. Therefore, using GADPH and ApMat gene regions confirmed the reliability and affordability of these markers to differentiate between species of C. gloeosporioides complex. Although C. siamense has been previously reported on Coffea arabica and many host species, this is the first report of C. siamense causing anthracnose on coffee in Saudi Arabia. This was also the first report of C. aeschynomenes on coffee in Saudi Arabia and worldwide. In addition, the two novel species; C. saudianum and C. coffeae-arabicae were new additions to the Colletotrichum species causing anthracnose on coffee in Saudi Arabia and worldwide. Furthermore, the dominance of C. saudianum makes it an appropriate model for addressing questions of population structure and dispersal at broad geographical and landscape level. Hence, additional collections from coffee growing regions across the southwest of Saudi Arabia would therefore aid us characterize the population structure of this important pathogen and to confirm whether this species is indeed the dominant Colletotrichum species.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9070705/s1, Table S1: Source, origin and date of collection of the 27 isolates obtained in this study