Fungicide resistance in Colletotrichum fructicola and Colletotrichum siamense causing peach anthracnose in China.

Peach is one of the popular and economically important fruit crops in China. Peach cultivation is hampered due to attacks of anthracnose disease, causing significant economic losses. Colletotrichum fructicola and Colletotrichum siamense belong to the Colletotrichum gloeosporioides species complex and are considered major pathogens of peach anthracnose. Application of different groups of fungicides is a routine approach for controlling this disease. However, fungicide resistance is a significant drawback in managing peach anthracnose nowadays. In this study, 39 isolates of C. fructicola and 41 isolates of C. siamense were collected from different locations in various provinces in China. The sensitivity of C. fructicola and C. siamense to some commonly used fungicides, i.e., carbendazim, iprodione, fluopyram, and propiconazole, was determined. All the isolates of C. fructicola collected from Guangdong province showed high resistance to carbendazim, whereas isolates collected from Guizhou province were sensitive. In C. siamense, isolates collected from Hebei province showed moderate resistance, while those from Shandong province were sensitive to carbendazim. On the other hand, all the isolates of C. fructicola and C. siamense showed high resistance to the dicarboximide (DCF) fungicide iprodione and succinate dehydrogenase inhibitor (SDHI) fungicide fluopyram. However, they are all sensitive to the demethylation inhibitor (DMI) fungicide propiconazole. Positive cross-resistance was observed between carbendazim and benomyl as they are members of the same methyl benzimidazole carbamate (MBC) group. While no correlation of sensitivity was observed between different groups of fungicides. No significant differences were found in each fitness parameter between carbendazim-resistant and sensitive isolates in both species. Molecular characterization of the ß-tubulin 2 (TUB2) gene revealed that in C. fructicola, the E198A point mutation was the determinant for the high resistance to carbendazim, while the F200Y point mutation was linked with the moderate resistance to carbendazim in C. siamense. Based on the results of this study, DMI fungicides, e.g., propiconazole or prochloraz could be used to control peach anthracnose, especially at locations where the pathogens have already developed the resistance to carbendazim and other fungicides.

First Report of Colletotrichum siamense Causing Fruit Anthracnose on Pomegranate (Punica granatum) in China.

As a native crop in central Asia, pomegranate (Punica granatum L.) has been cultivated in China for more than 2000 years (Yuan et al. 2007). In August 2022, typical symptoms of anthracnose were observed on pomegranate fruitlets in the main cultivation area (34°22'36″N, 109°15'58″E) in Shaanxi Province, China. The disease incidence was approximately 10 to 15% in the field. The initial symptoms were slightly small, light, dark, sunken lesions with irregular, circular shapes. As the disease progressed, the necrotic lesions gradually expanded and merged, eventually leading to the abscission of fruits (Figure 1, A). The symptomatic lesion samples of the pomegranate were sterilized for 1.5 min in 75% ethanol and 2 min in 1% NaClO and rinsed for 2 min in sterile water three times. The sterilized samples were dried on sterile filter paper and placed on potato dextrose agar (PDA) media at 25 ℃ for 5 days. The mycelia of the isolate were white, cottony, and diffuse (Figure 1, B and C). The conidia were single-celled, smooth, aseptate, and cylindrical with slightly rounded ends, measured 13.5 to 17.5 µm long and 3.5 to 6.5 µm wide (mean 16.0 × 4.5 µm, n = 50) (Figure 1, D). These morphological characteristics were identical to those of Colletotrichum siamense (Weir et al. 2012; Cannon et al. 2012; Zhuo et al. 2022). For accurate molecular characterization of the fungus, genomic DNA was extracted from the hyphae of the two isolates using microorganism lysis buffer (Takara, Japan). The internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) regions were amplified and sequenced. All the sequences were submitted to GenBank with accession numbers OQ832556 (ITS), OQ848589 (GAPDH), OQ848590 (ACT), OQ848591 (CAL), and OQ986593 (TUB2). The isolates showed 99 to 100% identity with sequences of Colletotrichum siamense (100% with GAPDH, ACT, CAL, and TUB2; 99.81% with ITS). The morphology of the strain was studied, and multilocus (ITS, GAPDH, ACT, CAL, and TUB2) phylogenetic analysis was performed (Figure 2). Therefore, the causal pathogen was identified as C. siamense based on the results of morphological and molecular analyses. Pathogenicity assays were performed on pomegranate (cv. Lishanhong) fruits. A conidial suspension (1×106 conidia/mL) was sprayed onto 10 unwounded fruits to inoculate them as infected samples, and the controls were inoculated with a sterile water suspension. All the samples were maintained in an artificial climate box at 25 ± 2 ℃ with 70% relative humidity, and the photoperiod was set as 12:12 light:dark. After 5 to 7 days, anthracnose symptoms developed on the surface of the inoculated fruit, whereas the control fruits remained healthy. The diseased fruits exhibited brown necrotic lesions, whereas the upper surfaces of the control fruits remained asymptomatic. The morphological and molecular characteristics of the reisolated pathogen were identical to those of the original fungus isolated from the natural fruit. C. siamense has been reported to cause anthracnose in the southeastern United States (Xavier et al. 2019). The pathogen causing anthracnose on pomegranates has been reported to be Colletotrichum fructicola in China (Hu et al. 2023). To our knowledge, this is the first report of anthracnose on pomegranate fruits caused by C. siamense in China. This disease can directly affect the quality and yield of the fruit. Thus, information about the characteristics of this disease could provide a theoretical basis for its prevention and control.

First Report of Colletotrichum siamense Causing Anthracnose on Bixa orellana in China.

Annatto (Bixa orellana L.) is widely cultivated in China. Its seed is used as medicine and as an astringent antipyretic. Since 2019, anthracnose-type lesions have been observed on the annatto leaves in the field (about 30 hectares) in Zhanjiang (21˚18'12''N, 110˚17'22''E), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 3 ha). The early symptoms were yellow spots on the edge or tip of leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions (Supplemental Figure 1-A). Ten symptomatic leaves from 10 plants were sampled. The margins of the lesions were cut into 2 × 2 mm pieces and the surfaces were disinfected with 75% ethanol for 30 sec and 2% sodium hypochlorite for 60 sec. After that, pieces were rinsed thrice in sterile water, placed on potato dextrose agar(PDA) medium, and incubated at 28 ℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (BOC-1, BOC-2, and BOC-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. Besides, the three isolates were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University. Colonies on PDA were white to gray with cottony mycelia after incubating in the dark for 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 9.6 to 18.5 µm × 3.5 to 5.5 µm (n = 50). Appressoria were oval to irregular in shape and dark brown, and they measured 6 to 9 µm × 4.5 to 8 µm (n = 30) (Supplemental Figure 1-D, E and F). These morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates were amplified using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences were deposited in GenBank under nos. MZ047377-MZ047379 (ITS), MZ126934-MZ1269346 (GAPDH), MZ126904-MZ1269046 (CHS-1), and MZ126844-MZ1268446 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from ITS, GAPDH, CHS-1, and ACT sequences that clustered the three isolates with C. siamense (the type strain MFLU 090230), (Supplemental Figure 2). The pathogenicity of the three isolates was tested respectively in a greenhouse maintained at 25 to 29℃ and 80% relative humidity. Annatto seeding ( n =5, 2-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water.. The experient was repeated three times. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy (Supplemental Figure 1-G, and H). The same pathogen was re-isolated from all the inoculated leaves based on morphology and ITS analysis. C. siamense has been reported to cause anthracnose in a broad range of hosts (Weir et al. 2012; Wang et al. 2017; Liu et al. 2017; Zhuo et al. 2017 ), but not in B. orellana. To our knowledge, this is the first report of C. siamense causing anthracnose on B. orellana in China. Our study provides important reference information for controlling this disease.

First report of Colletotrichum siamense causing anthracnose in Oenocarpus bacaba in Brazil.

Bacaba (Oenocarpus bacaba Mart.) is a native palm tree from Brazilian Amazon and Cerrado biomes. This tree produces a small, rounded fruit with dark skin and approximately 1.5 mm thick pulp, extensively utilized for palm heart extraction, juices, and jellies (De Cól et al. 2021). However, several diseases can adversely impact fruit yield and quality. During the 2021 growing season, anthracnose symptoms were observed in Bacaba fruits, with a disease incidence of 58% in fruits collected from the Abreulândia (9°37'15″ S, 49°9'3″ W) and Gurupi (12°25'46" S; 49°16'42" W) municipalities in Tocantins state, Brazil. A total of 198 fruits exhibiting anthracnose symptoms, characterized by deep necrotic spots, were collected. In the laboratory, symptomatic fruits had their external surfaces sterilized for 30 seconds in 70% ethanol, 1 min in 1.5% NaOCl, and then rinsed with sterile distilled water. Sterilized pieces of the fruit tissue were transferred to PDA medium and incubated for 7 days at 28 ºC with a 12 h photoperiod. After this period, two isolates were obtained from the colonies and were identified both macroscopically and microscopically as Colletotrichum sp. The colonies grown at PDA showed a white to grey cottony mycelia, with straight and fusiform conidia, ranging from 14.0 to 21.0 (mean value of 15.8 ± 1.8) µm in length and 4.0 to 7.0 (mean value of 5.5 ± 0.7) µm in width, (n = 50). For species identification, the intergenic spacer between DNA lyase, mating-type locus MAT1-2-1 (APN2/MAT-IGS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and ß-tubulin (TUB) loci were amplified and sequenced. Resulting sequences were deposited in GenBank (OR333843, OR333844, OR333845 and OR333846). BLAST analysis of the partial APN2/MAT-IGS (99%), GAPDH (99,48%), GS (99,32%) and TUB (99,48%) sequences showed highly similarity to C. siamense isolates (IIFT223 and CBS130147). Maximum likelihood multilocus analysis placed the isolate UFTC16 within the C. siamense clade with 98% bootstrap support, clearly assigning the isolate to this species. Morphological features were consistent with the description of C. siamense (Prihastuti et al., 2009). Inoculation of Bacaba fruits and seedlings was conducted to confirm pathogenicity. The surface of uninjured Bacaba fruits was inoculated with two drops (20 µL) of conidial suspension (106 conidia mL-1). The same methodology was adopted to placed healthy leaves of 35-day-old seedlings grown in plastic tubes. Two drops of sterile distilled water were inoculated on nonwounded healthy fruits and seedlings as a negative control. The fruits and seedlings were incubated for five days in a controlled chamber at 28 °C, 70-80% humidity and a "12-h photoperiod". The experiment was conducted with five replicates (five fruits and five seedlings inoculated per isolate) and repeated once. Typical symptoms of anthracnose were observed in the fruits and leaves of Bacaba seedlings five days after inoculation. No symptoms were observed in the negative control. The pathogen was reisolated from symptomatic fruits and leaves, showing similar morphological characteristics as the original isolate, fulfilling Koch's postulates. The identification of C. siamense as the causal agent of Bacaba anthracnose helps in the diagnosis and disease control strategies of the disease. Colletotrichum siamense is a cosmopolitan species and easily found in cultivated and non-cultivated species (Batista et al. 2023). However, to the best of our knowledge, this is the first report of C. siamense causing anthracnose on Bacaba.

Rapid Detection of Colletotrichum siamense from Infected Tea Plants Using Filter-Disc DNA Extraction and Loop-Mediated Isothermal Amplification.

The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/µl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.

First report of Colletotrichum siamense causing fruit anthracnose on Acer fabric in China.

Acer fabric Hance is an evergreen tree widely cultivated in China for its ornamental value (Liu et al. 2021). In August 2021, serious fruit anthracnose, with brown to black irregular sunken lesions, occurred on A. fabric plants at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E) in Nanchang, Jiangxi province, China. On average, 25% of the fruit per individual tree was affected. Small spots initially formed along the edge of the fruit and gradually expanded into dark brown spots, and eventually the diseased fruit withered. Small pieces (4 × 4 mm) from the affected fruits were surface sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Liao et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, AFG-3, AFG-7, and AFG-12, were used for morphological studies and phylogenetic analyses. Colonies on the PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture under incubation at 25°C in the dark for 7 days. Conidia were single-celled, hyaline, cylindrical, clavate, and measured 12.6-17.3 × 4.2-5.6 µm (14.2 ± 0.9 × 4.5 ± 0.3 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.5-9.2 × 4.5-6.7 µm (7.2 ± 0.2 × 5.4 ± 0.5 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, OQ184035 - OQ184037; ACT, OQ196109 - OQ196111; GAPDH, OQ196115 - OQ196117; TUB2, OQ196121 - OQ196123; CHS-1, OQ196112 - OQ196114; CAL, OQ196118 - OQ196120). A maximum likelihood and Bayesian posterior probability analysis using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed AFG-3, AFG-7, and AFG-12 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity tests were conducted on six 9-year-old A. fabric plants. Healthy fruits were wounded with a sterile needle and inoculated by placing a drop on the surface of wounded tissue (106 conidia/mL; 20 µL) prepared from the three isolates. The conidial suspension of each isolate was inoculated onto three fruit per plant. Another nine fruit on three plants inoculated with sterile water served as the control (Wan et al. 2022). All the inoculated fruit were covered with plastic bags to keep the humidity for two days. All the inoculated fruit showed dark brown spots similar to those symptoms observed on naturally infected fruit on campus, whereas control fruit were asymptomatic. C. siamense was reisolated from the inoculated fruit. The pathogen was previously reported to cause fruit anthracnose on Carya illinoinensis (Zhuo et al. 2022), Chili Pepper (May et al. 2021), and Salix babylonica (Zhang et al. 2023). This study confirmed that C. siamense also causes anthracnose on fruit of A. fabric. This work contributes to a better understanding of the etiology of fruit anthracnose on A. fabric in south China and helps develop effective control strategies.

Glucanases and Chitinases in Mangifera indica: Identification, Classification, Phylogeny, and Expression Analysis of Defense Genes against Colletotrichum spp.

Plant glucanases and chitinases are defense proteins that participate in pathogenesis; however, very little is known about the glucanase (GLUC) and chitinase (CHIT) gene families in mango. Some mango cultivars are of great economic importance and can be affected by anthracnose, a postharvest disease caused by fungi of the genus Colletotrichum spp. This study identified and characterized 23 putative glucanases and 16 chitinases in the mango genome cv. Tommy Atkins. We used phylogenetic analyses to classify the glucanases into three subclasses (A, B, and C) and the chitinases into four classes (I, II, IV, and V). Information on the salicylic, jasmonic acid, and ethylene pathways was obtained by analyzing the cis-elements of the GLUC and CHIT class I and IV gene promoters. The expression profile of GLUC, CHIT class I, and CHIT class IV genes in mango cv. Ataulfo inoculated with two Colletotrichum spp. revealed different profile expression related to these fungi's level of virulence. In general, this study provides the basis for the functional validation of these target genes with which the regulatory mechanisms used by glucanases and chitinases as defense proteins in mango can be elucidated.

Identification of gene modules and hub genes associated with Colletotrichum siamense infection in mango using weighted gene co-expression network analysis.

Colletotrichum siamense is a hemibiotrophic ascomycetous fungus responsible for mango anthracnose. The key genes involved in C. siamense infection remained largely unknown. In this study, we conducted weighted gene co-expression network analysis (WGCNA) of RNA-seq data to mine key genes involved in Colletotrichum siamense-mango interactions. Gene modules of Turquoise and Salmon, containing 1039 and 139 respectively, were associated with C. siamense infection, which were conducted for further analysis. GO enrichment analysis revealed that protein synthesis, organonitrogen compound biosynthetic and metabolic process, and endoplasmic reticulum-related genes were associated with C. siamense infection. A total of 568 proteins had homologs in the PHI database, 370 of which were related to virulence. The hub genes in each module were identified, which were annotated as O-methyltransferase (Salmon) and Clock-controlled protein 6 (Turquoise). A total of 24 proteins exhibited characteristics of SCRPs. By using transient expression in Nicotiana benthamiana, the SCRPs of XM_036637681.1 could inhibit programmed cell death (PCD) that induced by BAX (BCL-2-associated X protein), suggesting that it may play important roles in C. siamense infection. A mango-C. siamense co-expression network was constructed, and the mango gene of XM_044632979.1 (auxin-induced protein 15A-like) was positively associated with 5 SCRPs. These findings help to deepen the current understanding of necrotrophic stage in C. siamense infection.

Polymorphisms at amino acid positions 85 and 86 in succinate dehydrogenase subunit C of Colletotrichum siamense: Implications for fitness and intrinsic sensitivity to SDHI fungicides.

Among succinate dehydrogenase inhibiter (SDHI) fungicides, penthiopyrad and benzovindiflupyr particularly inhibit Colletotrichum. Studying SDH amino acid polymorphism in Colletotrichum, along with its fungicide binding sites, is key to understanding their mechanisms of action. This study explores the SDH amino acid polymorphisms in Colletotrichum siamense strains from rubber trees in China and their interaction with SDHI fungicides, specifically penthiopyrad and benzovindiflupyr. Sequencing revealed most polymorphisms were in the SDHC subunit, particularly at positions 85 and 86, which are key to penthiopyrad resistance. Among 33 isolates, 33.3 % exhibited a substitution at position 85, and 9 % at position 86. A strain with W85L and T86N substitutions in SDHC showed reduced SDH activity, ATP content, mycelial growth, and virulence, and decreased sensitivity to penthiopyrad but not benzovindiflupyr. Molecular docking with Alphafold2 modeling suggested distinct binding modes of the two fungicides to C. siamense SDH. These findings underscore the importance of SDHC polymorphisms in C. siamense's fitness and sensitivity to SDHIs, enhancing our understanding of pathogen-SDHI interactions and aiding the development of novel SDHI fungicides.

Gcc1 homologs regulate growth, oxidative stress, conidiation and appressorium formation in Colletotrichum siamense and Colletotrichum graminicola.

The Zn2Cys6 transcription factor is a fungal-specific zinc finger protein, which plays an important role in regulating growth, development and pathogenicity of pathogenic fungi. In this study, we characterized two Zn2Cys6 transcription factors, CsGcc1 and CgrGcc1 in Colletotrichum siamense and C. graminicola, respectively, which are homologous to Gcc1 in Magnaporthe oryzae. Both CsGcc1 and CgrGcc1 contain a typical GAL4 DNA-binding domain. Deletion of CsGCC1 or CgrGCC1 decreased the growth rate and lowered the tolerance to H2O2. In addition, disrupting CsGCC1 reduced conidial yield and lowered the germination rate and appressorium formation rate of C. siamense. Cellophane assays showed that deletion of CsGCC1 also weakened the penetration ability of appressoria. In C. graminicola, CgrGcc1 did not affect the production and germination of oval conidia, but its deletion significantly decreased the yield of the falcate conidium, and led to abnormal appressorium formation. In terms of pathogenicity, CsGcc1 slightly reduced the virulence of C. siamense, while deleting CgrGcc1 did not affect virulence of C. graminicola. In conclusion, the Zn2Cys6 transcription factors CsGcc1 and CgrGcc1 are involved in the regulation of vegetative growth, oxidative stress, conidial/falcate conidial production and appressorium formation in C. siamense and C. graminicola.

First Report of Colletotrichum siamense Causing Anthracnose on Syzygium grijsii in China.

Syzygium grijsii is an evergreen shrub belonging to the family Myrtaceae, and widely cultivated in southern China as an ornamental medicinal plant. In May 2022, anthracnose symptoms were observed on leaves of S. grijsii planted in a nursery (N22°55'46″, E108°22'11″) in Nanning, Guangxi Province, China. More than 30% of leaves were infected. Initially, irregular brown spots (1 to 2 mm in diameter) formed on the leaves, with a slight depression in the center, then expanded into large, dark-brown lesions. In severe infections, lesions coalesced and covered the entire leaf, causing wilt and fall off the plant. To identify the pathogen, 30 diseased leaves were collected from five plants. Leaf tissues (5 × 5 mm) were cut from the infected margins, surface sterilized (75% ethanol 10 s, 2% NaClO 5 min, rinsed three times with sterile water), then placed on potato dextrose agar (PDA), and incubated at 28℃ in darkness. After 5 days, 16 fungal isolates with similar morphology were obtained from 30 plated tissues. Colonies on PDA were abundant with grayish-white fluffy mycelia, and yellowish-white on the back. Conidia were one-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, blunt at the ends, and ranged from 11.35 to 22.14 × 4.88 to 7.67 µm (n=100). Morphological characteristics of the isolates were similar to the descriptions of Colletotrichum sp. (Prihastuti et al. 2009). Five representative isolates (Cs34, Cs31, Cs32, Cs33 and Cs35), which were preserved in the Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, were selected for molecular identification. The ITS (Nos. OQ618199, OR539576 to OR539579), TUB2 (Nos. OQ630972, OR545076 to OR545079), ACT (Nos. OQ685919, OR545060 to OR545063), CHS-1 (Nos. OQ685917, OR545068 to OR545071), GAPDH (Nos. OQ685916, OR545072 to OR545075), and CAL (Nos. OQ685918, OR545064 to OR545067) sequences showed >99% identity to those of Colletotrichum siamense ex-type culture ICPM 18578 (Nos. JX010171, JX009924, JX009714 and JX009518) and strain C1315.2 (Nos. JX009865 and JX010404) in GenBank. Multigene phylogenetic analyses (ITS, TUB, ACT, CHS-1, GAPDH, and CAL) using the Maximum likelihood method indicated that the 5 isolates were clustered with C. siamense. To perform pathogenicity tests, three one-year-old healthy S. grijsii plants were inoculated with conidial suspension (1 × 106 conidia/ml) of isolate Cs34 by brushing gently with a soft paintbrush, each plant was inoculated with 3 leaves. The same number of plants were inoculated with sterile water as control, and pathogenicity tests were performed three times. All plants were kept in an artificial climatic box at 28℃, with a 90% humidity and a 12 h light/dark cycle. Similar symptoms to those of the field were observed on all inoculated leaves after 5 days, whereas controls remained symptomless. Reisolated fungi from the diseased leaves were confirmed to be C. siamense by morphology and molecular characterization, confirming Koch's postulates. C. siamense has been reported causing anthracnose on Crinum asiaticum (Khoo et al. 2022) in Malaysia, and Erythrina crista-galli in China (Li et al. 2021). To our knowledge, this is the first report of C. siamense causing anthracnose on S. grijsii in China. The results of pathogen identification provide crucial information for control strategies of the disease.

First report of anthracnose caused by Colletotrichum siamense on Illicium verum in China.

Aniseed (Illicium verum) is a woody spice tree that has been grown in China for a long time. Anthracnose is an important disease of aniseed, which can cause severe leaf drop. In Sep. 2020, severe anthracnose was observed in Shanglin (23°35'5"N, 108°19'51"E), Nanning, Guangxi in China, and the incidence was 85%. The symptoms at the early stage were small, round and watery, then became larger and gradually turned brown. The acervuli would appear at the later stage, and contain many conidia. Leaves with disease were randomly sampled from 10 plants, and were cut into small rectangular pieces of 0.5×1 cm, and disinfected with 75% alcohol 1 min, with 0.1% HgCl2 3 min. After washing with sterile water 3 times, they were placed onto potato dextrose agar (PDA) medium and incubated at 25°C for 5 days. The average colony growth rate was 11.85 mm/d in 7 days. The colony was white or light gray in the initial stage, with dense aerial mycelium, and the central mycelium of the colony was dark grey in the later stage. Conidia were colorless, single spore, smooth, cylindrical, both ends obtuse, with an average size of 14.95 ± 0.97 µm × 5.46 ± 0.44 µm (n = 100). The conidial appressorium was oval or club-shaped, brown, margin intact, with an average size of 7.83 ± 1.21 µm × 5.82 ± 0.58µm (n = 50). Three strains GXNN02, GXNN03 and GXNN05 were selected for further study. Primer pairs T1/ßt2b, ACT512/ACT783, GDF/GDR, CHS1-79F / CHS1-354R and ITS1/ITS4 (Weir et al. 2012) were used to amplify tubulin (TUB), actin (ACT), 3-phosphate glyceraldehyde dehydrogenase (GAPDH), chitinase (CHS1) and the internal transcribed spacers of rDNA (ITS) respectively. BLASTn searches showed that the TUB (ON619861-63 ), ACT (ON619852-54), GAPDH (ON619855-57), CHS1 (ON619858-60) and ITS (ON573028-30) sequences had the highest similarity to Colletotrichum siamense with up to 99% (699/702, 676/679, 699/702) identity for TUB (JX010404.1); 99% (281/282, 253/254, 249/250) identity for ACT (JX009518.1); 99% (275/277, 275/277, 239/241) identity for GAPDH (JX009924.1); 99% (296/299, 296/299, 259/262) identity for CHS1 (JX009865.1); up to 99% (527/530, 485/487, 527/530) identity for ITS (JX010171.1) of ex-type ICMP 18578. A ML tree was constructed by combining 5 sequenced loci, and three isolates clustered in the C. siamense clade with 94% bootstrap support. Therefore, combined with the morphological characteristics, the pathogens were identified as C. siamense. In a pathogenicity test, these three isolates were tested on 9 healthy aniseed seedlings with at least 10 leaves, and 3 seedlings as control. The leaves were surface disinfected with 75% alcohol, and then wiped with sterilized water three times. Holes were made near the edge of the leaves and were sprayed with conidial solution (6×106 spores/mL) in test groups, and use sterilized water as control. Then the leaves were sealed inside a plastic bag for 48 h to retain moisture. Brown spot and black acervuli, similar to the symptoms in the field, were observed on the leaves in test groups within 10-15 days. No symptoms were observed on the negative control leaves. The pathogens were reisolated from the treated infected leaves and were identified as C. siamense, thus fulfilling Koch's postulates. The pathogenicity test was confirmed by repeating in triplicate. The isolation frequency of C. siamense in our samples was 82.50%. To our knowledge, this is the first report of C. siamense in China. Further research on the occurrence of the disease will help prevent the spread of the disease.

First report of Colletotrichum siamense causing leaf spot on Celtis sinensis in China.

Celtis sinensis Pers., a deciduous tree, is widely cultivated in China for its ornamental value (Yang et al. 2022). In July 2020, leaf spot symptoms were observed on Ce. sinensis plants at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E) in Nanchang city, Jiangxi province, China. The disease incidence was estimated to be above 15%. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown, eventually developing large irregular lesions. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced and sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, JPS-4, JPS-9, and JPS-13 were used for morphological studies and phylogenetic analyses. Colonies on PDA medium of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.2 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.8 µm (7.6 ± 0.1 × 5.4 ± 0.2 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), ß-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, ON207804 - ON207806; ACT, ON239113 - ON239115; GAPDH, ON239122 - ON239124; TUB2, ON239125 - ON239127; CHS-1, ON239119 - ON239121; CAL, ON239116 - ON239118). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed JPS-4, JPS-9, and JPS-13 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. To confirm pathogenicity, nine 6-year-old Ce. sinensis plants (three leaves each, n=27) grown outdoors were pin-pricked with a sterile needle and inoculated with 100 µL spore suspension per leaf (106 conidia per mL). Another 27 healthy leaves were inoculated with sterile water as the control. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment for 2 days. The experiment was repeated three times. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. Colletotrichum siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. Colletotrichum siamense can cause leaf diseases in a variety of hosts, including Allamanda cathartica (Huang et al. 2022), Osmanthus fragrans (Liu et al. 2022), and Crinum asiaticum (Khoo et al. 2022). To our knowledge, this is the first report of C. siamense causing leaf spots on Ce. sinensis worldwide. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Colletotrichum siamense as a causal agent of anthracnose on wax apple (Syzygium samarangense) in China.

Wax apple (Syzygium samarangense) is an important fruit tree widely cultivated in China. Yield losses are usually serious due to different diseases among which anthracnose (Colletotrichum spp.) is one of the most damaging (He et al, 2019). This disease was found in Yunnan, China and an average incidence of 56.7% diseased leaves was recorded in21 orchards surveyed in July2021. The disease lesions on leaves were circular, angular or oval (7.2-15.6 mm), with whitish center and brown outer area surrounded by a yellow halo; irregular spots or blight areas formed later. It can also infect fruits forming pale-brown, circular and sunken spots before harvest and rot of stored fruits. Diseased leaves were sampled from orchards in Ximeng (N117.78oE39.89o) and Ninger (E101.04oN23.05o) counties of Yunnan for fungal isolation; three and five pure isolates were recovered from Ximeng (LWTJ1-LWTJ3) and Ninger (LB4-LB8) samples, respectively, by plating disinfested tissue (surface-sterilized with 2% NaClO3) on potato dextrose agar (PDA) followed by hyphal tip purification and incubation at 25oC. Two repeated tests following Koch's postulates were conducted to verify pathogenicity of the eight isolates. In each test, three healthy seedlings per isolate were sprayed with conidia suspenson (2.26×105cfu/mL) until runoff from leaves while control plants were sprayed with sterile water. The plants were kept in the dark at RH100 for 24 h in a black box and then in a growth chamber (28oC, RH>90% and lighting 12h/d). Detached fruits were inoculated with mycelial discs on the puncture-wound surface. Anthracnose symptoms developed on all seedlings and fruits inoculated with LWTJ2 or LB4 isolates, which were re-isolated from lesions of inoculated leaf/fruit, completing Koch's postulates. Control plants were healthy and symptomless. LWTJ2 and LB4 isolates were morphologically the same: the colonies on PDA were circular, pale-white, with cottony surface and readily forming orange conidium masses. The hyphae were hyaline, septate, branched mostly in near right angles. The conidia were hyaline, one-celled, smooth-walled, cylindrical with round ends, 9.8-17.5 (av.13.8) µm×4.4-6.5 (5.6) µm. The teleomorph was not observed in culture or on orchard trees. The morphological characters were consistent with those of C. siamense described by Weir et al (2012). The internal transcribed spacer region (ITS) was amplified from the two isolates by PCR and sequenced (1990) and were 545 bp in length (OL963924 & OL413460). BLAST analysis showed that both were 100% identical and they shared 99.08% identity with C. siamense WZ-365 from the ITS region (MN856443).The Tub2 (788 bp, ON637119) and Cal (768 bp, ON622249) genes (Weir et al, 2012) of LB4 were also obtained and they shared closest identity (99.45% & 100%) with those of C. siamense WZ-365 as well. Phylogenetic tree (neighbor-joining) analysis of the concatenated sequence of ITS, Tub2 and Cal genes of LB4 and those of related Colletotrichum spp. showed that LB4 clustered IN the same end-branch with C. siamense ICMP18578 (Bootstrap sup. = 98%). Thus, C. siamense was identified as the pathogen of wax apple anthracnose in Yunnan. It caused anthracnose on other crops as oranges and cacao (Azad et al, 2020). Also, C. fructicola and C. syzygicola were identified as pathogens of wax apple anthracnose in Thailand (Al-Obaidi et al, 2017). To our knowledge, this is the first report of C. siamense causing wax apple anthracnose in China.

First report of Colletotrichum siamense causing leaf spot on Macropanax rosthornii in China.

Macropanax rosthornii (Harms) C. Y. Wu ex Hoo is an evergreen broadleaf species cultivated in subtropical China as an ornamental (Liang et al. 2015). In August 2020, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E), Jiangxi province, China. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown with reddish egdes, eventually developing large irregular lesions. The disease incidence was estimated at 45%. Leaf pieces (5 × 5 mm) from the lesion borders were surface disinfested in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Li et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Three representative single-spore isolates (DS-2, DS-3, and DS-5) were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white-to-gray with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.1 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.9 µm (7.7 ± 0.3 × 5.5 ± 0.2 µm, n=100). Morphological features were similar to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, calmodulin (CAL), actin (ACT), ß-tubulin 2 (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, CL1/CL2, ACT-512F/ACT-783R, T1/Bt2b, GDF/GDR and CHS-79F/CHS-354R (Weir et al. 2012), respectively. Sequences were deposited in GenBank under nos. OL895315 - OL895316 (ITS), OL830190 - OL830192 (ACT), OL830181 - OL830183 (GAPDH), OL830178 - OL830180 (TUB2), OL830184 - OL830186 (CHS-1), and OL830187 - OL830189 (CAL). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed DS-2, DS-3, and DS-5 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity of the three isolates was verified on six 5-year-old Macropanax rosthornii plants, which were grown in the field. Three healthy leaves per plant were wounded using a sterile needle (Φ=0.5 mm) and inoculated with a 20-µL conidial suspension per leaf (106 conidia/mL). Another six control plants were treated with sterile water. Eighteen leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain humidity for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 8 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, including Liriodendron chinense × tulipifera (Zhu et al. 2019), Salix matsudana (Zhang et al. 2021), Carya illinoinensis (Zhuo et al. 2023). However, this is the first report of C. siamense infecting Macropanax rosthornii in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Leaf Spot Caused by Colletotrichum siamense, C. fructicola, and C. aeschynomenes on Ixora chinensis in Guangxi, China.

Ixora chinensis Lam., an important ornamental flower, has become more and more popular in the southwest and southeast regions of China for its bright and abundant flowers (Li et al. 2019). In March 2022, 100% I. chinensis plants showed typical anthracnose symptoms on leaf in Nanning, Guangxi, China (108°22' N, 22°48' E). The central areas of lesions were grayish white with small black particles arranged in a wheel pattern, and the edges of lesions were light red to brown. Three diseased leaf samples were collected from three gardens, respectively. 5×5 mm tissues were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on PDA at 25°C under 12/12 h light/darkness. Eighty-three morphologically similar colonies were observed on PDA after 5 days, and eight isolates G1-3, G2-1, G3-3, W-1, W-2, LCH2-1, LCH3-3, and LCH4-1 were selected for further study. Genomic DNA of these isolates were extracted from 7-day-old mycelia. Primer pairs ITS1/ITS4, GDF1/GDR1, T1/ßt2b, CHS Ⅰ-79F/CHS Ⅰ-354R, CL1/CL2, ACT-512F/ACT-783R, and MAT1-2-1/apn2 were used to amplify ITS loci and GAPDH, CHS-Ⅰ, CAL, ACT, ApMAT genes, respectively (Yang et al. 2009; Silva et al. 2012; Liu et al. 2015). Sequences have been deposited in GenBank (ITS: OQ771884 to OQ771891, GAPDH: OQ759576 to OQ759583, TUB2: OQ759584 to OQ759591, CHS-1: OQ759568 to OQ759575, CAL: OQ759560 to OQ759567, ACT: OQ759552 to OQ759559, ApMat: OQ759544 to OQ759551). Phylogenetic analysis was performed with raxmlGUI v.2.0.0. based on combined sequences of ITS, GAPDH, TUB2, CHS-1, CAL, ACT, and ApMAT using maximum parsimony analysis. The results revealed that isolates G2-1 and W-2 were clustered with Collectrichum fructicola, G3-3, W-1, G1-3, LCH2-1, and LCH3-3 were clustered with C. siamense, and LCH4-1 was clustered with C. aeschynomenes. Three representative isolates W-2, G3-3, and LCH4-1 were selected for morphology and pathogenicity observation. On PDA, the colonies of three isolates presented white to grey cottony mycelia，from the margin to the center, W-2 was white, grey, and light gray, G3-3 showed light gray, white, and grey, LCH4-1 was white and light gray, respectively. Conidia were all hyaline, one-celled, cylindrical, and straight. The conidial sizes of W-2, G3-3, and LCH4-1 were 11.03 to 17.53 × 4.93 to 8.42 µm (n=100), 10.63 to 19.06 × 3.73 to 6.92 µm (n=100), and 11.61 to 20.39 × 3.65 to 6.67 µm (n=100), respectively. Pathogenicity tests of three isolates were conducted on leaves of 1-year-old I. chinensis plants with and without wounds, three plants for each treatment, and five leaves inoculated for each plant. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site. Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton, and all plants were bagged and placed in the greenhouse to maintain humidity at 25℃. After 10 days, all wounded and inoculated leaves showed leaf spot, whereas unwounded and control leaves remained asymptomatic. Koch's postulates were fulfilled by re-isolating the causal agents from diseased leaves. C. siamense and C. aeschynomenes could cause anthracnose of I. chinensis in China (Liu et al. 2016, Li et al. 2021). However, to our knowledge, this is the first report of C. fructicola infecting I. chinensis in China. This study may provide reference for further epidemiological study and prevention of anthracnose on I. chinensis.

Colletotrichum siamense leaf blight/spot: Characterization of a newly identified disease on Cinnamomum tamala.

Cinnamomum tamala (bay leaf) is widely used for culinary and medicinal purposes in South Asia. A leaf blight/spot disease was first discovered on nearly 90% of C. tamala plants with a mean severity of 48% to 74.4% in Gazipur and Bogura, Bangladesh, in 2019. The present study identified and characterized the causal organism and formulated the optimum growth conditions and effective fungicides for the chemical control of the pathogen. The characteristic symptoms on the infected leaves appeared circular to oval reddish-brown spots with raised margins and often developed in tear-stain patterns. Severe infection of C. tamala sapling resulted in dieback symptoms with leaf defoliation. A fungus with floccose, dense, white colonies with well-differentiated acervuli was isolated from the infected leaves. Combined cultural, morphological, and molecular characteristics identified the pathogen as Colletotrichum siamense. Inculcating healthy leaves and 1-year-old saplings of C. tamala with a conidial suspension of the fungus reproduced the same symptoms observed in the bay leaf orchard. The highest mycelial growth was recorded on V-8 Juice Agar media, while the maximum radial mycelial growth and level of sporulation of the fungus were significantly higher in incubation temperature 30°C. Fungicide trials showed that carbendazim 50 WP, azoxystrobin, mancozeb, and trifloxystrobin, either singly or in combination, successfully reduced fungal mycelial growth in vitro. Therefore, disease management strategies should be opted to halt the further spread of this issue. To our knowledge, this is the first study to document the incidence of Colletotrichum leaf blight on C. tamala in Bangladesh and even in the world.

The Plasma Membrane H+ ATPase CsPMA2 Regulates Lipid Droplet Formation, Appressorial Development and Virulence in Colletotrichum siamense.

Plasma membrane H+-ATPases (PMAs) play an important role in the pathogenicity of pathogenic fungi. Lipid droplets are important storage sites for neutral lipids in fungal conidia and hyphae and can be used by plant pathogenic fungi for infection. However, the relationship between plasma membrane H+-ATPase, lipid droplets and virulence remains unclear. Here, we characterized a plasma membrane H+-ATPase, CsPMA2, that plays a key role in lipid droplet formation, appresorial development and virulence in C. siamense. Deletion of CsPMA2 impaired C. siamense conidial size, conidial germination, appressorial development and virulence but did not affect hyphal growth. ΔCsPMA2 increased the sensitivity of C. siamense to phytic acid and oxalic acid. CsPMA2 was localized to lipids on the plasma membrane and intracellular membrane. Deletion of CsPMA2 significantly inhibited the accumulation of lipid droplets and significantly affected the contents of some species of lipids, including 12 species with decreased lipid contents and 3 species with increased lipid contents. Furthermore, low pH can inhibit CsPMA2 expression and lipid droplet accumulation. Overall, our data revealed that the plasma membrane H+-ATPase CsPMA2 is involved in the regulation of lipid droplet formation and affects appressorial development and virulence in C. siamense.

CsAtf1, a bZIP transcription factor, is involved in fludioxonil sensitivity and virulence in the rubber tree anthracnose fungus Colletotrichum siamense.

In phytopathogenic fungi, the HOG MAPK pathway has roles in osmoregulation, fungicide sensitivity, and other processes. The ATF1/CREB-activating transcription factor Atf1 is a regulator that functions downstream of the HOG MAPK pathway. Here, we identified a gene, designated CsAtf1, that encodes a bZIP transcription factor in Colletotrichum siamense, which is the main pathogen that causes Colletotrichum leaf fall disease in rubber trees in China. CsAtf1 localizes to the nucleus. Its mRNA expression correlates positively with that of CsPbs2 and CsHog1 in the HOG MAPK pathway in response to activator (anisomycin), inhibitor (SB203580) and fludioxonil treatments. The CsAtf1 deletion mutant showed slightly retarded mycelial growth, small conidia, slow spore germination, and abnormal appressorium formation. This mutant showed the increased spore germination rate after fludioxonil treatment and more resistance to the fungicide fludioxonil than did the wild-type fungus. However, unlike deletion of Pbs2 or Hog1, which resulted in greater sensitivity to osmotic stress, the CsAtf1 deletion induced slightly increased resistance to osmotic stress and the cell wall stress response. The ΔCsAtf1 strain also exhibited significantly reduced virulence on rubber tree leaves. These data revealed that CsAtf1 plays a key role in the regulation of fludioxonil sensitivity and in pathogenicity regulation in C. siamense.

First Report of Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde causing anthracnose disease in Santalum album Linn. in India.

Indian sandalwood (Santalum album), valued for its medicinal properties, is an indigenous species of India. Circular or irregular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the centre of the lesion were observed on leaves of sandalwood seedlings in a nursery located in Karnataka, with a disease incidence of 75% (n = 100 investigated plants) during July 2020. The disease prevailed in monsoon followed by winter season (July 2020 - January 2021); summer was less supportive for the disease incidence. As the disease progressed, lesions expanded and merged, causing necrosis of the whole leaf. Isolation of the pathogen involved excision of small sections of diseased tissues from the lesions followed by surface sterilization with 70% ethanol for 30 s and in 1% NaClO for 1 min. Sections were rinsed in sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Ten isolates of Colletotrichum ssp. were obtained with an isolation frequency = 10/12×100 = 83%. One representative single-spore isolate (CSSA-1) was used for further study. Initially, pure cultures exhibited a white mycelium which later turned gray with time, and had orange conidial masses in a concentric ring pattern with the aggregation of black acervuli at the center of the culture Conidia were single celled, hyaline, and cylindrical having smooth rounded ends and the size ranged from 12.6 to 18.5 µm in length, and 3.5 to 5.6 µm in width (n = 100). The morphological characteristics were in agreement with the species description of fungi in the Colletotrichum gloeosporioides species complex (Weir et al. 2012). To confirm the species designation of the isolate CSSA-1, a multilocus phylogenetic analysis was performed using six genomic loci (Weir et al. 2012; Marin-Felix et al. 2017). The internal transcribed spacer (ITS) region of rDNA and a partial sequence of the beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT), and glutamine synthetase (GS) genes were amplified using ITS1/ITS4, BT2F/BT4R, GAPDHF/R, 79F/354R, 512F/783R and GSF/GSR primers, respectively. The ITS (OK254122), TUB2 (OL462863), GAPDH (OL462859), CHS-1 (OL462860), actin (OL462861), and glutamine synthetase (OL961822) sequences of representative isolate CSSA-1 showed 99 to 100% identity with sequences MZ148628, MK967339, MN525882, MW192791, MT263504, MF111030, MH370542 and KX578767, respectively to the holotype isolate of Colletotrichum siamense (Prihastuti et al. 2009). The sequences were analysed with representative sequences of Colletotrichum and a multilocus Bayesian inference phylogenetic tree with ITS-GAPDH-ACT-CHS1-GS-TUB2 concatenated data sets (concatenated with Sequence Matrix v.1.8 (Vaidya et al. 2011)) was constructed using Beast version 1.8.4 to confirm the isolate identification (Drummond et al. 2012; Hyde et al. 2014; Weir et al. 2012). Isolate CSSA-1 clustered with C. siamense isolates. To complete Koch's postulates, for the characterized isolate CSSA-1, a pathogenicity test was conducted on 3-month-old sandalwood seedlings by spore spray inoculation. Ten plants were inoculated with a conidial suspension (106 conidia/ml) and control plants inoculated with sterilized water then kept in a glass house at 25°C and >85% relative humidity with a 12-h photoperiod. Humidity was maintained by spraying the plants with water in the morning and evening to enhance the infection. Typical symptoms of anthracnose disease similar to naturally infected leaves were observed, which included circular pale yellow lesions surrounded by a purple halo with prominent pinhead sized black fruiting bodies at the center of the lesion 7 days after inoculation, while the control plants remained unaffected. The pathogen was reisolated from infected leaves and its identity was confirmed as C. siamense based on morphological characteristics. Previously, C. siamense was identified causing disease on chili in India (Gunjan and Shenoy, 2014), but to our knowledge this is the first report of leaf anthracnose caused by C. siamense on Indian sandalwood in India or globally. This study documents crucial information, paving way for epidemiologic studies and design of control strategies to combat this newly emerging disease.