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

Several <i>Colletotrichum</i> species are able to cause anthracnose disease in coffee (<i>Coffea arabica</i> 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 <i>Colletotrichum</i>-like fungi were obtained using a spore suspension method. The taxonomic placements of <i>Colletotrichum</i>-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 <i>Colletotrichum</i> 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 <i>C. gloeosporioides</i> complex, while the remaining two isolates were assigned to the <i>C. boninense</i> complex. Six species were recognized, four of them, <i>C. aeschynomenes, C. siamense, C. phyllanthi,</i> and <i>C. karstii</i>, had been previously described. Based on molecular analyses and morphological examination comparisons, <i>C. saudianum</i> and <i>C. coffeae-arabicae</i> represent novel members within the <i>C. gloeosporioides</i> complex. Pathogenicity investigation confirmed that the <i>Colletotrichum</i> 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 <i>C. aeschynomenes, C. siamense, C. karstii, C. phyllanthi, C. saudianum,</i> and <i>C. coffeae-arabicae</i> to cause anthracnose on coffee in Saudi Arabia.