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.

Synergistic Biocontrol and Growth Promotion in Strawberries by Co-Cultured Trichoderma harzianum TW21990 and Burkholderia vietnamiensis B418.

This study aimed to investigate the efficiency of the secondary metabolites (SMs) produced by a co-culture of Trichoderma harzianum TW21990 and Burkholderia vietnamiensis B418 in the control of Colletotrichum siamense CM9. A fermentation filtrate of B418 + TW21990 co-culture (BT21) produced a notable increase in the inhibition rate of CM9 compared to those of TW21990 and B418 monocultures, which reached 91.40% and 80.46% on PDA plates and strawberry leaves, respectively. The BT21 fermentation broth exhibited high control efficiency on strawberry root rot of 68.95% in a pot experiment, which was higher than that in the monocultures and fluazinam treatment. In addition, BT21 treatment promoted strawberry root development, improved antioxidative enzyme activities in the leaves and roots, and enhanced the total chlorophyll content of the strawberry leaves. UHPLC-MS/MS analysis of fermentation filtrates was performed to elucidate SM variations, revealing 478 and 795 metabolites in BT21 co-culture in positive and negative ion modes, respectively. The metabolomic profiles suggested abundant SMs with antagonistic capabilities and growth-promoting effects: 3-(propan-2-yl)-octahydropyrrolo [1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Val)), 3-[(4-hydroxyphenyl)methyl]-octahydropyrrolo[1,2-a]pyrazine-1,4-dione (cyclo(L-Pro-L-Tyr)), 3-indoleacetic acid (IAA), 2-hydroxycinnamic acid, 4-aminobutyric acid (GABA), bafilomycin B1, and DL-indole-3-lactic acid (ILA) were significantly enhanced in the co-culture. Overall, this study demonstrates that a co-culture strategy is efficient for inducing bioactive SMs in T. harzianum and B. vietnamiensis, which could be exploited as a novel approach for developing biocontrol consortia.

Development and Comparison of Visual LAMP and LAMP-TaqMan Assays for Colletotrichum siamense.

Strawberry anthracnose caused by Colletotrichum spp. has resulted in significant losses in strawberry production worldwide. Strawberry anthracnose occurs mainly at the seedling and early planting stages, and Colletotrichum siamense is the main pathogen in North China, where mycelia, anamorphic nuclei, and conidia produced in the soil are the main sources of infection. The detection of pathogens in soil is crucial for predicting the prevalence of anthracnose. In this study, a visualized loop-mediated isothermal amplification (LAMP) assay and a loop-mediated isothermal amplification method combined with a TaqMan probe (LAMP-TaqMan) assay were developed for the ß-tubulin sequence of C. siamense. Both methods can detect Colletotrichum siamense genomic DNA at very low concentrations (104 copies/g) in soil, while both the visualized LAMP and LAMP-TaqMan assays exhibited a detection limit of 50 copies/µL, surpassing the sensitivity of conventional PCR and qPCR techniques, and both methods showed high specificity for C. siamense. The two methods were compared: LAMP-TaqMan exhibited enhanced specificity due to the incorporation of fluorescent molecular beacons, while visualized LAMP solely necessitated uncomplicated incubation at a constant temperature, with the results determined by the color change; therefore, the requirements for the instrument are relatively straightforward and user-friendly. In conclusion, both assays will help monitor populations of C. siamense in China and control strawberry anthracnose in the field.

The C2H2 Transcription Factor Con7 Regulates Vegetative Growth, Cell Wall Integrity, Oxidative Stress, Asexual Sporulation, Appressorium and Hyphopodium Formation, and Pathogenicity in Colletotrichum graminicola and Colletotrichum siamense.

The Colletotrichum genus is listed as one of the top 10 important plant pathogens, causing significant economic losses worldwide. The C2H2 zinc finger protein serves as a crucial transcription factor regulating growth and development in fungi. In this study, we identified two C2H2 transcription factors, CgrCon7 and CsCon7, in Colletotrichum graminicola and Colletotrichum siamense, as the orthologs of Con7p in Magnaporthe oryzae. Both CgrCon7 and CsCon7 have a typical C2H2 zinc finger domain and exhibit visible nuclear localization. Disrupting Cgrcon7 or Cscon7 led to a decreased growth rate, changes in cell wall integrity, and low tolerance to H2O2. Moreover, the deletion of Cgrcon7 or Cscon7 dramatically decreased conidial production, and their knockout mutants also lost the ability to produce appressoria and hyphopodia. Pathogenicity assays displayed that deleting Cgrcon7 or Cscon7 resulted in a complete loss of virulence. Transcriptome analysis showed that CgrCon7 and CsCon7 were involved in regulating many genes related to ROS detoxification, chitin synthesis, and cell wall degradation, etc. In conclusion, CgrCon7 and CsCon7 act as master transcription factors coordinating vegetative growth, oxidative stress response, cell wall integrity, asexual sporulation, appressorium formation, and pathogenicity in C. graminicola and C. siamense.

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 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.

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra.

Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.

The CsPbs2-interacting protein oxalate decarboxylase CsOxdC3 modulates morphosporogenesis, virulence, and fungicide resistance in Colletotrichum siamense.

The HOG MAPK pathway mediates diverse cellular and physiological processes, including osmoregulation and fungicide sensitivity, in phytopathogenic fungi. However, the molecular mechanisms underlying HOG MAPK pathway-associated stress homeostasis and pathophysiological developmental events are poorly understood. Here, we demonstrated that the oxalate decarboxylase CsOxdC3 in Colletotrichum siamense interacts with the protein kinase kinase CsPbs2, a component of the HOG MAPK pathway. The expression of the CsOxdC3 gene was significantly suppressed in response to phenylpyrrole and tebuconazole fungicide treatments, while that of CsPbs2 was upregulated by phenylpyrrole and not affected by tebuconazole. We showed that targeted gene deletion of CsOxdC3 suppressed mycelial growth, reduced conidial length, and triggered a marginal reduction in the sporulation characteristics of the ΔCsOxdC3 strains. Interestingly, the ΔCsOxdC3 strain was significantly sensitive to fungicides, including phenylpyrrole and tebuconazole, while the CsPbs2-defective strain was sensitive to tebuconazole but resistant to phenylpyrrole. Additionally, infection assessment revealed a significant reduction in the virulence of the ΔCsOxdC3 strains when inoculated on the leaves of rubber tree (Hevea brasiliensis). From these observations, we inferred that CsOxdC3 crucially modulates HOG MAPK pathway-dependent processes, including morphogenesis, stress homeostasis, fungicide resistance, and virulence, in C. siamense by facilitating direct physical interactions with CsPbs2. This study provides insights into the molecular regulators of the HOG MAPK pathway and underscores the potential of deploying OxdCs as potent targets for developing fungicides.

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 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.

Re-identification of Colletotrichum gloeosporioides Species Complex Isolates in Korea and Their Host Plants.

The Colletotrichum gloeosporioides species complex includes many phytopathogenic species, causing anthracnose disease on a wide range of host plants and appearing to be globally distributed. Seventy-one Colletotrichum isolates in the complex from different plants and geographic regions in Korea were preserved in the Korean Agricultural Culture Collection (KACC). Most of them had been identified based on hosts and morphological features, this could lead to inaccurate species names. Therefore, the KACC isolates were re-identified using DNA sequence analyses of six loci, comprising internal transcribed spacer, gapdh, chs-1, his3, act, and tub2 in this study. Based on the combined phylogenetic analysis, KACC strains were assigned to 12 known species and three new species candidates. The detected species are C. siamense (n = 20), C. fructicola (n = 19), C. gloeosporioides (n = 9), C. aenigma (n = 5), C. camelliae (n = 3), C. temperatum (n = 3), C. musae (n = 2), C. theobromicola (n = 2), C. viniferum (n = 2), C. alatae (n = 1), C. jiangxiense (n = 1), and C. yulongense (n = 1). Of these, C. jiangxiense, C. temperatum, C. theobromicola and C. yulongense are unrecorded species in Korea. Host plant comparisons showed that 27 fungus-host associations are newly reported in the country. However, plant-fungus interactions need to be investigated by pathogenicity tests.

A New Biocontrol Agent Bacillus velezensis SF334 against Rubber Tree Fungal Leaf Anthracnose and Its Genome Analysis of Versatile Plant Probiotic Traits.

Natural rubber is an important national strategic and industrial raw material. The leaf anthracnose of rubber trees caused by the Colletotrichum species is one of the important factors restricting the yields of natural rubber. In this study, we isolated and identified strain Bacillus velezensis SF334, which exhibited significant antagonistic activity against both C. australisinense and C. siamense, the dominant species of Colletotrichum causing rubber tree leaf anthracnose in the Hainan province of China, from a pool of 223 bacterial strains. The cell suspensions of SF334 had a significant prevention effect for the leaf anthracnose of rubber trees, with an efficacy of 79.67% against C. siamense and 71.8% against C. australisinense. We demonstrated that SF334 can lead to the lysis of C. australisinense and C. siamense mycelia by causing mycelial expansion, resulting in mycelial rupture and subsequent death. B. velezensis SF334 also harbors some plant probiotic traits, such as secreting siderophore, protease, cellulase, pectinase, and the auxin of indole-3-acetic acid (IAA), and it has broad-spectrum antifungal activity against some important plant pathogenic fungi. The genome combined with comparative genomic analyses indicated that SF334 possesses most genes of the central metabolic and gene clusters of secondary metabolites in B. velezensis strains. To our knowledge, this is the first time a Bacillus velezensis strain has been reported as a promising biocontrol agent against the leaf anthracnose of rubber trees caused by C. siamense and C. australisinense. The results suggest that B. velezensis could be a potential candidate agent for the leaf anthracnose of rubber trees.

The fatty acid 2-hydroxylase CsSCS7 is a key hyphal growth factor and potential control target in Colletotrichum siamense.

SCS7 is a fatty acid 2-hydroxylase required for the synthesis of inositol phosphorylceramide but is not essential for normal growth in Saccharomyces cerevisiae. Here, we demonstrate that the Colletotrichum siamense SCS7 homolog CsSCS7 plays a key role in hyphal growth. The CsSCS7 deletion mutant showed strong hyphal growth inhibition, small conidia, and marginally reduced sporulation and also resulted in a sharp reduction in the full virulence and increasing the fungicide sensitivity. The three protein domains (a cytochrome b5 domain, a transmembrane domain, and a hydroxylase domain) are important to CsSCS7 protein function in hyphal growth. The fatty acid assay results revealed that the CsSCS7 gene is important for balancing the contents of multiple mid-long- and short-chain fatty acids. Additionally, the retarded growth and virulence of C. siamense ΔCsSCS7 can be recovered partly by the reintroduction of homologous sequences from Magnaporthe oryzae and Fusarium graminearum but not SCS7 of S. cerevisiae. In addition, the spraying of C. siamense with naked CsSCS7-double-stranded RNA (dsRNAs), which leads to RNAi, increases the inhibition of hyphal growth and slightly decreases disease lesions. Then, we used nano material Mg-Al-layered double hydroxide as carriers to deliver dsRNA, which significantly enhanced the control effect of dsRNA, and the lesion area was obviously reduced. These data indicated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.IMPORTANCECsSCS7, which is homologous to yeast fatty acid 2-hydroxylase SCS7, was confirmed to play a key role in the hyphal growth of Colletotrichum siamense and affect its virulence. The CsSCS7 gene is involved in the synthesis and metabolism of fatty acids. Homologs from the filamentous fungi Magnaporthe oryzae and Fusarium graminearum can recover the retarded growth and virulence of C. siamense ΔCsSCS7. The spraying of double-stranded RNAs targeting CsSCS7 can inhibit hyphal growth and reduce the disease lesion area to some extent. After using nano material Mg-Al layered double hydroxide as carrier, the inhibition rates were significantly increased. We demonstrated that CsSCS7 is an important factor for hyphal growth and affects virulence and may be a potential control target in C. siamense and even in filamentous plant pathogenic fungi.

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.

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.

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.

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.

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.

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.