Characterization and Fungicide Sensitivity of Colletotrichum spp. from Capsicum peppers in South Korea.

Capsicum peppers, peppers from plants of the genus Capsicum (family Solanaceae), are widely cultivated in South Korea, where annual production was 92,756 tons in 2021, 54.4% higher than that of the previous year. Occurring throughout the production cycle, anthracnose is a major disease limiting commercial Capsicum pepper production worldwide, including in South Korea. This study investigates the diversity and pathogenicity of Colletotrichum species responsible for Capsicum pepper anthracnose in Gyeongbuk, South Korea, focusing on disease incidence and symptomatology in the field and the identification, morphological characteristics, pathogenicity, and fungicide sensitivity of the causative species. Disease incidence ranged from 30 to 50%, with samples categorized into three distinct symptom types, aiding accurate field diagnosis. Phylogenetic analysis classified 41 isolates into six species in the acutatum, gloeosporioides, and truncatum species complexes, revealing significant genetic diversity. Morphological characterization supported these identifications, providing a comprehensive profiling. Pathogenicity tests confirmed that all identified species induced typical anthracnose lesions, with lesion size variations suggesting differential aggressiveness. Temperature significantly influenced mycelial growth, with optimal growth between 20 and 26°C, and C. truncatum demonstrating high-temperature tolerance.In vitro fungicide sensitivity tests showed variable responses, with tebuconazole being generally effective. These findings underscore the need for species-specific fungicide recommendations and highlight the importance of continuous monitoring of Colletotrichum species. Future research should explore the molecular mechanisms of pathogenicity, host specificity, and fungicide resistance, integrating these findings with breeding programs to develop resistant pepper varieties. This study provides critical insights for effective anthracnose management in pepper cultivation and future research directions.

BDPapayaLeaf: A dataset of papaya leaf for disease detection, classification, and analysis.

Papaya is a popular vegetable and fruit in both developing and developed countries. Nonetheless, Bangladesh's agricultural landscape is significantly influenced by papaya cultivation. However, disease is a common impediment to papaya productivity, adversely affecting papaya quality and yield and leading to substantial economic losses for farmers. Research suggests that computer-aided disease diagnosis and machine learning (ML) models can improve papaya production by detecting and classifying diseases. In this line, a dataset of papaya is required to diagnose the disease. Moreover, like many other fruits, papaya disease may vary from country to country. Therefore, the country-based papaya disease dataset is required. In this study, a papaya dataset is collected from Dhaka, Bangladesh. This dataset contains 2159 original images from five classes, including the healthy control class and four papaya leaf diseases: Anthracnose, Bacterial Spot, Curl, and Ring spot. Besides the original images, the dataset contains 210 annotated data for each of the five classes. The dataset contains two types of data: the whole image and the annotated image. The image will interest data scientists who apply disease detection through a convolutional neural network (CNN) and its variants. Furthermore, the annotated images, such as You Only Look Once (YOLO), U-Net, Mask R-CNN, and Single Shot Detection (SSD), will be helpful for semantic segmentation. Since firm-applicable AI devices and mobile and web applications are in demand, the dataset collected in this study will offer multiple options for integrating ML models into AI devices. In countries with weather and climate similar to Bangladesh, data scientists may use their dataset in that context.

The yeast Wickerhamomyces anomalus acts as a predator of the olive anthracnose-causing fungi, Colletotrichum nymphaeae, C. godetiae, and C. gloeosporioides.

Olive tree anthracnose is caused by infection with Colletotrichum fungi, which in Portugal are mostly C. nymphaeae, C. godetiae, and C. gloeosporioides s.s. Severe economic losses are caused by this disease that would benefit from a greener and more efficient alternative to the present agrochemical methods. Yeasts are serious candidates for pre-harvest/in field biocontrol of fungal infections. This work identified the yeast Wickerhamomyces anomalus as a strong antagonizer of the three fungi and studied in vitro this ability and its associated mechanisms. Antagonism was shown to not depend on the secretion of volatile compounds (VOCs), or siderophores or any other agar-diffusible compound, including hydrolytic enzymes. Rather, it occurred mostly in a cell-to-cell contact dependent manner. This was devised through detailed microscopic assessment of yeast-fungus cocultures. This showed that W. anomalus antagonism of the three Colletotrichum proceeded through (i) the adhesion of yeast cells to the phytopathogen hyphae, (ii) the secretion of a viscous extracellular matrix, and (iii) the emptying of the hyphae. Yeasts ultimately putatively feed on hyphal contents, which is supported by light microscopy observation of MB and PI co-culture-stained samples. Accordingly, numerous W. anomalus cells were observed packing inside C. godetiae emptied hyphae. This behaviour can be considered microbial predation and classified as necrotrophic mycoparasitism, more explicitly in the case of C. godetiae. The results support the prospect of future application of W. anomalus as a living biofungicide/BCA in the preharvest control of olive anthracnose.

Anthracnose Caused by Colletotrichum spaethianum on Polygonatum odoratum var. pluriflorum in Korea.

Polygonatum odoratum var. pluriflorum (Miq.) Ohwi (Asparagaceae), commonly known as Lesser Solomon's seal, is a perennial herbaceous plant widely distributed in the temperate regions of the Northern Hemisphere. It is known for tea and contains various pharmacologically active compounds (Chang et al. 2007). In September 2021, previously unseen anthracnose was encountered on Polygonatum odoratum var. pluriflorum in Chuncheon (37°45'07''N; 127°47'45''E) and Wanju (35°38'47''N; 127°31'16''E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The disease symptoms begin with leaf yellowing (chlorosis) along the margins, progressing toward the central part of the leaf. As the disease progressed, brown necrotic lesions developed, covered with small, dark dots, indicating the presence of conidiomata of Colletotrichum. The disease often led to leaf blight, resulting in concentric lesions of a periodic and concentric arrangement of conidiomata, forming circular patterns within the affected tissue. It expanded rapidly during the rainy season producing water-soaked lesions but nearly slowed down during the dry season, leaving the plant with a ragged appearance. Monoconidial isolates were obtained from five fresh samples collected in Wanju, of which a representative isolate was deposited in the Korean Agricultural Culture Collection (KACC410442), and the dried specimen was housed at the herbarium of Jeonbuk National University (JBNU0135). Morphological characteristics of the fungus were examined with fresh and naturally infected leaves. Setae (n = 30) were dark brown to nearly blackish, 45 to 172 um long, 4.1 to 6.1 µm wide at the base, and becoming narrower upwards, apex pointed, 2 to 4-septate. Conidia were 1-celled, sometimes uniseptate before germination, slightly curved, with an acute or rounded apex and a more or less truncate base, and 11.4 to 23.2 × 3.1 to 4.2 µm. Appressoria were single or in groups, dark brown, lobate, irregularly shaped, and 6.0 to 11.9 × 7.0 to 14.8 µm. Two-week-old colonies grown on PDA at 25 ℃ reached 30-35 mm in diam., initially white, turning gray with age, with cottony aerial mycelia. These morphological characteristics were in good agreement with those of C. spaethianum (Damm et al. 2009; Liu et al. 2020). To confirm the morphology-based determination, genomic DNA was extracted from KACC410442, and multi-loci sequences of the internal transcribed spacer region (ITS) rDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin (actA), chitin synthase (CHS1), histone (HIS3) and tubulin (TUB2) genes were determined, as outlined by Cannon et al. (2012) and Damm et al. (2009). The resulting sequences obtained in this study were registered to GenBank (PP739190 for ITS, PP741985 for GAPDH, PP741986 for actA, PP741987 for CHS1, PP741988 for HIS3, and PP741989 for TUB2) and compared with other sequences on GenBank using the BLASTn search tool. The results showed 100% identity to other C. spaethianum sequences (e.g., MT611068 for ITS, MN400659 for actA, OP965007 for CHS1, OR060963 for GAPDH, PP480643 for HIS3, and OQ613727 for TUB2). In a maximum parsimony tree, the Korean isolate formed a well-supported clade (100% bootstrap value) with reference sequences of C. spaethianum. Pathogenicity was tested by spraying a conidial suspension (1×104 conidia/ml) from a two-week-old culture onto ten leaves of three potted plants, without wounding. Ten water-sprayed leaves were served as controls. The plants were maintained in a dew chamber and later moved to a greenhouse at 25 to 30 °C. After two weeks, all inoculated plants developed anthracnose symptoms, while the control plants remained symptomless. The fungus isolated from the inoculated plants was morphologically identical to that initially observed, fulfilling Koch's postulates. C. spaethianum has been known on various plant species, but mostly on the Asparagaceae, including Anemarrhena (Okorley et al. 2019), Hosta (Damm et al. 2009; Cheon and Jeon 2016), and Polygonatum (Liu et al. 2020; Ma et al. 2021). To our knowledge, this is the first report of anthracnose caused by C. spaethianum on P. odoratum var. pluriflorum in Korea, highlighting the continuous need for disease management and control in the cultivation of this plant.

Dual-RNA-sequencing to elucidate the interactions between sorghum and Colletotrichum sublineola.

In warm and humid regions, the productivity of sorghum is significantly limited by the fungal hemibiotrophic pathogen Colletotrichum sublineola, the causal agent of anthracnose, a problematic disease of sorghum (Sorghum bicolor (L.) Moench) that can result in grain and biomass yield losses of up to 50%. Despite available genomic resources of both the host and fungal pathogen, the molecular basis of sorghum-C. sublineola interactions are poorly understood. By employing a dual-RNA sequencing approach, the molecular crosstalk between sorghum and C. sublineola can be elucidated. In this study, we examined the transcriptomes of four resistant sorghum accessions from the sorghum association panel (SAP) at varying time points post-infection with C. sublineola. Approximately 0.3% and 93% of the reads mapped to the genomes of C. sublineola and Sorghum bicolor, respectively. Expression profiling of in vitro versus in planta C. sublineola at 1-, 3-, and 5-days post-infection (dpi) indicated that genes encoding secreted candidate effectors, carbohydrate-active enzymes (CAZymes), and membrane transporters increased in expression during the transition from the biotrophic to the necrotrophic phase (3 dpi). The hallmark of the pathogen-associated molecular pattern (PAMP)-triggered immunity in sorghum includes the production of reactive oxygen species (ROS) and phytoalexins. The majority of effector candidates secreted by C. sublineola were predicted to be localized in the host apoplast, where they could interfere with the PAMP-triggered immunity response, specifically in the host ROS signaling pathway. The genes encoding critical molecular factors influencing pathogenicity identified in this study are a useful resource for subsequent genetic experiments aimed at validating their contributions to pathogen virulence. This comprehensive study not only provides a better understanding of the biology of C. sublineola but also supports the long-term goal of developing resistant sorghum cultivars.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

First report of Colletotrichum liaoningense causing anthracnose on winter squash in Korea.

Winter squash (Cucurbita maxima) is rich in vitamins C and B6 and is also a source of beta-carotene, a provitamin A carotenoid. About 13,000 tons have been produced annually in South Korea over the past 10 years. In the summer of 2022, severe rot was observed in winter squash for sale at a wholesale market in Jinju, South Korea, with approximately 10% of the 500 squashes observed affected. White fungal hyphae and dark orange spore masses were observed on the surface of the decayed squash. To isolate the causal agents, symptomatic tissues (3 × 3 mm) between diseased and healthy tissues per squash from 3 diseased squashes were excised, disinfested with 1% sodium hypochlorite for 20 s and 70% ethanol for 10 s, washed twice in sterilized distilled water, dried on sterilized filter paper, transferred to water agar, and incubated at 25°C for 2 days. Agar blocks (3 mm2) containing fungal colonies were transferred to potato dextrose agar (PDA) plates and incubated at 25°C until fungal colonies grew. Three isolates (GNU F137a‒c) with similar morphology were subcultured using the single-spore method. In PDA, the colonies looked like gray cotton when viewed from the front, were pale orange from the back, and numerous small black sclerotia-like grains could be observed on both sides. Setae were pale to medium brown, verrucose, 40-120 µm long, and 3-6 septated. Conidiophores were hyaline to pale brown, smooth-walled, septate, branched, and up to 45 µm long. Conidia were hyaline, smooth walled, aseptate, straight, cylindrical, the apex and base rounded, and 14-18 × 5-7 µm (n = 30). Appressoria were single, brown, aseptate, ellipsoidal to irregular in outline, with crenate margins, and 3.5-5 × 3-5 µm (n = 30). The morphological features of the fungal isolates matched descriptions of Colletotrichum species. To confirm the identity of the isolated fungus, genomic DNA of all three isolates was extracted using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Baltics, UAB). The internal transcribed spacers (ITS) of the ribosomal RNA gene region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT), and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4, GDF/GDR, CHS-79F/CHS-354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R, and T1/T2, respectively. The sequences were deposited in GenBank (acc. nos., PP504320 and PP555649-PP555653). Concatenated sequences of the six genes obtained from isolates GNU F137a‒c and ex-types from each accepted taxon in previous studies were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11. The fungus isolated from winter squash was in the same clade as C. liaoningense. Therefore, the isolates were identified as C. liaoningense. For pathogenicity tests, three winter squash were wounded with a sterilized needle and inoculated with each isolate by injecting 100 µl conidial suspension (105 conidia/ml). Control squash were injected with sterilized distilled water. All treated squash were incubated at 25°C in the dark. The test was performed three times. All inoculated winter squash reproduced symptoms within 15 days, whereas the control squash were symptomless. The morphological characteristics and ITS sequence of the re-isolated strain matched those of the inoculated strain. To the best of our knowledge, this is the first report of fruit rot of winter squash in Korea and is even the first report on C. liaoningense in Korea. This disease is considered a post-harvest disease because no cases have yet been discovered in the field in Korea. This report will facilitate epidemiological research and the development of effective disease control strategies.

Biocontrol potential of Bacillus velezensis HG-8-2 against postharvest anthracnose on chili pepper caused by Colletotrichum scovillei.

Anthracnose caused by Colletotrichum scovillei is a significant disease of pepper, including in postharvest stage. Bacillus species represent a potential microbial resource for controlling postharvest plant diseases. Here, a strain HG-8-2 was obtained and identified as Bacillus velezensis through morphological, biochemical, physiological, and molecular analyses. The culture filtrate showed highly antifungal activity against C. scovillei both in vitro and on pepper fruit. Crude lipopeptide extracts, which had excellent stability, could effectively inhibit mycelial growth of C. scovillei with an EC50 value of 28.48 ± 1.45 µg mL-1 and inhibited conidial germination. Pretreatment with the extracts reduced the incidence and lesion size of postharvest anthracnose on pepper fruit. Analysis using propidium iodide staining, malondialdehyde content detection and scanning electron microscope observation suggested that the crude lipopeptide extracts harbored antifungal activity by damaging cell membranes and mycelial structures. The RNA-seq analysis conducted on C. scovillei samples treated with the extracts, as compared to untreated samples, revealed significant alterations in the expression of multiple genes involved in protein biosynthesis. Overall, these results demonstrated that B. velezensis HG-8-2 and its crude lipopeptide extracts exhibit highly antagonistic ability against C. scovillei, thereby offering an effective biological agent for the control of anthracnose in pepper fruit.

Plant-derived citronellol can significantly disrupt cell wall integrity maintenance of Colletotrichum camelliae.

Anthracnose, a fungal disease, commonly infects tea plants and severely impacts the yield and quality of tea. One method for controlling anthracnose is the application of citronellol, a plant extract that exhibits broad-spectrum antimicrobial activity. Herein, the physiological and biochemical mechanism by which citronellol controls anthracnose caused by Colletotrichum camelliae was investigated. Citronellol exhibited excellent antifungal activity based on direct and indirect mycelial growth inhibition assays, with EC50 values of 76.88 mg/L and 29.79 µL/L air, respectively. Citronellol also exhibited good control effects on C. camelliae in semi-isolated leaf experiments. Optical and scanning electron microscopy revealed that citronellol caused C. camelliae mycelia to thin, fracture, fold and deform. Transmission electron microscopy revealed that the mycelial cell walls collapsed inward and separated, and the organelles became blurred after treatment with citronellol. The sensitivity of C. camelliae to calcofluor white staining was significantly enhanced by citronellol, while PI staining showed minimal fluorescence, and the relative conductivity of mycelia were not significantly different. Under citronellol treatment, the expression levels of ß-1,3-glucanase, chitin synthase, and chitin deacetylase-related genes were significantly decreased, while the expression levels of chitinase genes were increased, leading to lower chitinase activity and increased ß-1,3-glucanase activity. Therefore, citronellol disrupted the cell wall integrity of C. camelliae and inhibited normal mycelial growth.

Cercosporamide, a polyketide-derived fungal metabolite, serves as an antifungal agent against phytopathogenic fungi.

An endophytic fungus, Phoma sp. NG-25, produces a set of structurally related polyketides including cercosporamide, phomodione, and usnic acid, among which, cercosporamide has been reported to have strong antifungal and anticancer activities. In this study, Phoma sp. NG-25 was grown in seven growth media to determine the optimal culture condition conducive for cercosporamide production. Cercosporamide production peaked on the eighteenth day of incubation in beef peptone dextrose (BPD) broth media. The cercosporamide titer reached to an average of 77.5 µg/mL in BPD. Paper disk diffusion assay revealed that culture filtrate containing cercosporamide as a major constituent inhibited the growth of taxonomically diverse plant pathogens, including ascomycetous, basidiomycetous, and oomycete fungi. Cercosporamide exhibited strong antifungal activities against two pepper anthracnose pathogens, Colletotrichum gloeosporioides and C. scovillei with EC50 values of 3.8 and 7.0 µg/mL, respectively. This study suggests the potential application of cercosporamide as an effective antifungal agent in controlling anthracnose in pepper.

Transcription factor CpWRKY50 enhances anthracnose resistance by promoting jasmonic acid signaling in papaya.

Colletotrichum brevisporum is an important fungal pathogen that causes anthracnose and has led to serious postharvest losses of papaya (Carica papaya L.) fruit in recent years. WRKY transcription factors play vital roles in regulating plant resistance to pathogens, but their functions in papaya anthracnose resistance need further exploration. In this study, we identified a WRKY transcription factor, CpWRKY50, which belongs to the WRKY IIc subfamily. During infection with C. brevisporum, expression of CpWRKY50 in anthracnose-resistant papaya cultivars was significantly higher than that in susceptible cultivars. CpWRKY50 was induced by methyl jasmonate, and CpWRKY50 localized in the nucleus. In yeast, full-length CpWRKY50 had transactivation activity, but CpWRKY50 variants truncated at the N or C termini did not. CpWRKY50 positively regulated papaya resistance to C. brevisporum, as demonstrated by transient overexpression of CpWRKY50 in papaya and heterologous expression of CpWRKY50 in tomato. Moreover, endogenous jasmonic acid (JA) and JA-isoleucine levels in the fruits of transgenic tomato OE lines were higher than in wild type both before and after inoculation with C. brevisporum, indicating that increased CpWRKY50 expression promotes JA accumulation. Furthermore, our results revealed CpWRKY50 directly binds to W-box motifs (TTGACC) in the promoters of two JA signaling-related genes, CpMYC2 and pathogenesis-related 4 CpPR4, thereby activating their expression. Our data support that CpWRKY50 positively regulates anthracnose resistance in papaya by promoting JA signaling. These results broaden our understanding of papaya disease resistance mechanisms and will facilitate the genetic improvement of papaya through molecular breeding.

First Report of Chili Anthracnose Caused by Colletotrichum sojae in South Korea.

Chili (Capsicum annuum L.) is an economically important crop worldwide, valued for its culinary uses. In South Korea, anthracnose caused by Colletotrichum spp. including C. truncatum, C. gloeosporioides, C. coccodes, C. acutatum, and C. scovillei incurs on substantial economic loss (Kim et al. 2008; Oo and Oh 2020). In August 2022, somewhat different types of symptoms that was not typical on chilli fruits were observed in a field in Yereonggwang (GPS: 35.2579° N, 126.4742° E), South Korea. The disease symptoms appeared as sunken, necrotic lesions with dense black spore masses forming in concentric rings. The estimated disease incidence the 0.2 ha field showing up to 1% of fruits affected. To isolate the pathogen, six symptomatic chilli fruits were collected. Small pieces (5 mm²) were cut from the margins of the lesions, surface-sterilized in 70% ethanol for 30 sec, followed by 1% sodium hypochlorite for 1 minute, and then rinsed three times in sterile distilled water. The tissue pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C in the dark. After 3 to 5 days, emerging fungal colonies were sub-cultured to obtain pure isolates. A total of five isolates were obtained and initially identified as Colletotrichum spp. based on morphological characteristics. Seven-day old colonies were initially white, turning light orange with age on PDA. Setae (observed on lesion) were dark brown, verruculose and septate. Conidia were cylindrical, hyaline, and measured 14.8 to 19.9 × 4.2 to 6.5 µm (mean 16.7 × 5.6 µm, n = 70) in size; appressoria were brown to dark brown and irregularly shaped. These morphological characteristics of the isolates agree with those reported for the morphology of C. sojae by Damm et al. (2019). To confirm the identity of the isolates, DNA was extracted and specific gene regions were amplified and sequenced using the following primer sets: ITS (ITS1 and ITS4), GAPDH (GDF1 and GDR1), ACT (ACT-512F and ACT-783R), TUB (T1 and Bt2b), HIS3 (CYLH3F and CYLH3R), and CHS-1 (CHS-79F and CHS-345R). The resulting sequences were deposited in the NCBI GenBank with accession numbers (LC830742 to LC830766). Maximum likelihood phylogenetic analysis using combine sequences of ITS, GAPDH, ACT, TUB, HIS3 and CHS-1 in MEGA X confirmed the isolates as C. sojae, marking the first report of this pathogen on chilli in South Korea, previously known to infect soybean. Pathogenicity tests were conducted on wound and nonwounded healthy and mature-green chili fruits (cv. Bicksita) to confirm the pathogenicity of the isolated C. sojae. The fruits were surface-sterilized using 70% ethanol and then rinsed with sterile distilled water. The fruits were wounded using a sterile needle to facilitate infection. A conidial suspension (1x106 conidia/mL) was prepared from 7-day-old PDA cultures. Each fruit was inoculated by placing a 10 µL drop of the conidial suspension onto the wounded and nonwounded sites (4 to 5) of the wound and unwound fruits, respectively. Control fruits were inoculated with sterile water. A total of 40 fruits per treatment were used and the experiment repeated twice. The fruits were placed in plastic box lined with moist paper towels to maintain high humidity and incubated at 25°C. Anthracnose symptoms developed on the inoculated fruits within 7 days, while control and unwounded fruits remained symptom-free. Colletotrichum sojae was successfully reisolated from the symptomatic fruits, fulfilling Koch's postulates and confirming its role as the causal agent of the disease. Colletotrichum sojae is known to infect Fabaceae species worldwide such as Glycine max, Medicago sativa, Phaseolus vulgaris, Atractylodes ovata and Vigna unguiculata (Damm et al. 2019; Talhinhas and Baroncelli 2021), Atractylodes ovata in South Korea (Hassan et al. 2021) and chili pepper in China (Zhanget al. 2023). The first report of C. sojae causing chili anthracnose in South Korea represents a new challenge for chili growers. Integrated disease management strategies need to be developed and implemented to mitigate its impact.

Colletotrichum species (Glomerellales, Glomerellaceae) causing walnut anthracnose in China.

Colletotrichum species can function as plant pathogens, saprobes or endophytes on a wide variety of plant hosts and are considered amongst the ten most significant genera of plant pathogens globally. China contributes almost half the walnut production in the world. However, Colletotrichum species occurring on walnut remain largely unresolved in China. To explore the Colletotrichum species found on walnut in China, 470 walnut fruit or leaf samples with anthracnose were collected from 14 main walnut-producing regions across seven provinces. A total of 165 Colletotrichum strains were isolated from these samples. The Colletotrichum isolates were identified, based on morphological characteristics and sequence analyses of ACT, CHS-1, GAPDH, ITS and TUB2. Twelve species, including 11 known Colletotrichum species (C.boninense, C.citrulli, C.fioriniae, C.fructicola, C.godetiae, C.juglandicola, C.karsti, C.mengyinense, C.pandanicola, C.peakense and C.siamense) and a novel species (C.chinensis sp. nov.) were identified. The species distribution revealed regional prevalence as follows: C.mengyinense was the most dominant species in Gansu, C.mengyinense and C.siamense in Shandong, C.chinensis in Beijing, C.pandanicola in Shaanxi and C.godetiae in Yunnan. Colletotrichumsiamense was the sole species isolated in Sichuan and Xinjiang Provinces. Koch's postulates were fulfilled, demonstrating that all 12 species cause anthracnose on walnut. This is the first report of C.boninense, C.citrulli and C.karsti as pathogens of walnut anthracnose worldwide.

The response of root-zone soil bacterial community, metabolites, and soil properties of Sanyeqing medicinal plant varieties to anthracnose disease in reclaimed land, China.

Objectives: To enhance the utilization of reclaimed land, Sanyeqing (SYQ) has been extensively cultivated in Zhejiang province, China. However, the prevalence of anthracnose has significantly hindered SYQ growth, emerging as a primary obstacle to its production. This study aimed to elucidate SYQ's responses to anthracnose in reclaimed land environments by comprehensively analyzing root-zone bacterial community structure, metabolites, and soil properties. Methods: The experiment was conducted on reclaimed land in Chun'an, China. In order to evaluate the responses of SYQ to anthracnose, the fresh and dry weight of SYQ tubes, the soil properties, the high-throughput sequencing, and metabolomics assay were carried out. Results: Significant differences were observed between an anthracnose-resistant variety (A201714) and an anthracnose-susceptibile variety (B201301). Fresh and dry weight increased 131.53 % and 144.82 % for A201714 compared to B201301.Lacibacterium (39.85 %), Gp6 (21.83 %), Gp5 (21.49 %), and Sphingomonas (18.84 %) were more prevalent, whereas Gp3 (22.71 %), WPS-1 (18.88 %), Gp4 (15.60 %), Subdivision3 (14.70 %), Chryseolinea (14.37 %), and Nitrospira (0.76 %) were less prevalent in A201714 than B201301. A total of 24 bacterial biomarkers were detected in all soil samples, while the network suggests a more stable soil bacterial community in A201714 than in B201301. Eight differentially expressed metabolites (DEMs) that belonged to lipids and lipid-like molecules, organic acids and derivatives, benzenoids, nucleosides, nucleotides, and analogues were found between two soil samples, and all these eight DEMs were downregulated in A201714 and had a strong correlation with 12 genera of bacteria. Moreover, the data from the redundancy analysis indicated that the main variables affecting changes in the bacterial communities were pH, available phosphorus (AP), available potassium (AK), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN). Conclusion: This research offers new insights into the SYQ response to anthracnose in reclaimed land and provides valuable recommendations for the high-quality SYQ cultivation and production.

Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of CGSC in strawberry seedlings. This method targets the ß-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA (ssDNA). This method shows high sensitivity (ten copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1-2 min). Furthermore, the Cas12aVIP assay successfully detected CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.

Complete Annotated Genome Assembly of Flax Pathogen Colletotrichum lini.

Colletotrichum lini is a fungal pathogen of flax that can cause significant yield and quality losses. In this work, we obtained the first complete annotated genome assembly of the highly virulent C. lini strain #394-2. The nuclear genome consisted of ten core and two accessory chromosomes and had a length of 53.7 Mb. The mitochondrial genome was 39.1 kb. The assembly was obtained by the Canu-Racon ×2-Medaka-Polca algorithm using Oxford Nanopore Technologies and Illumina data. As a result of the annotation with the Illumina RNA-Seq data, 12,449 genes were identified. Potential signaling proteins were tested for effector functions and 550 effector proteins were predicted using EffectorP. The visualization of the effector protein localization revealed that the presence of effector proteins was associated with repeat-rich regions. A comparison of the genomic structure of C. lini with chromosome-level and complete assemblies of the genus Colletotrichum representatives revealed that the genomes of Colletotrichum species differed by the presence of chromosomal rearrangements. The obtained assembly expands the knowledge of the genomic structure of Colletotrichum species and provides the basis for further studies of C. lini, which will help to understand the virulence mechanisms and protect flax from anthracnose.

Characterization and Pathogenicity of Colletotrichum truncatum Causing Hylocereus undatus Anthracnose through the Changes of Cell Wall-Degrading Enzymes and Components in Fruits.

Anthracnose is one of the destructive diseases of pitaya that seriously affects the plant growth and fruit quality and causes significant yield and economic losses worldwide. However, information regarding the species of pathogens that cause anthracnose in pitaya (Hylocereus undatus) fruits in Gansu Province, China, and its pathogenic mechanism is unknown. Thus, the purposes of our present study were to identify the species of pathogens causing H. undatus fruits anthracnose based on the morphological and molecular characteristics and determine its pathogenic mechanism by physiological and biochemical methods. In our present study, forty-six isolates were isolated from the collected samples of diseased H. undatus fruits and classified as three types (named as H-1, H-2, and H-3), according to the colony and conidium morphological characteristics. The isolation frequencies of H-1, H-2, and H-3 types were 63.04%, 21.74%, and 15.22%, respectively. The representative single-spore isolate of HLGTJ-1 in H-1 type has significant pathogenicity, and finally we identified Colletotrichum truncatum as the pathogen based on the morphological characteristics as well as multi-locus sequence analysis. Moreover, the H. undatus fruits inoculated with C. truncatum had a significantly increased activity of cell wall-degrading enzymes (CWDEs) cellulase (Cx), ß-glucosidase (ß-Glu), polygalacturonase (PG), and pectin methylgalacturonase (PMG), while having a decreased level of cell wall components of original pectin and cellulose in comparison to control. The average increased activities of Cx, ß-Glu, PG, and PMG were 30.73%, 40.40%, 51.55%, and 32.23% from day 0 to 6 after inoculation, respectively. In contrast, the average decreased contents of original pectin and cellulose were 1.82% and 16.47%, respectively, whereas the average increased soluble pectin content was 38.31% in comparison to control. Our results indicate that C. truncatum infection increased the activities of CWDEs in H. undatus fruits to disassemble their cell wall components, finally leading to the fruits' decay and deterioration. Thus, our findings will provide significant evidence in the controlling of pitaya anthracnose in the future.

Biocontrol of Colletotrichum fructicola in the Postharvest Banana Fruit Using the Siderophore-Producing Strain BX1.

Banana anthracnose, caused by Colletotrichum fructicola, significantly reduced the postharvest fruit quality. Employing biocontrol strategies offers a sustainable approach to enhance agricultural practices. The Burkholderia sp. strain BX1 hinders the growth and appressorium formation of C. fructicola, and its sterile filtrate lowers the anthracnose incidence while preserving the fruit quality. Scanning electron microscopy and genomic analyses confirmed BX1 as Burkholderia pyrrocinia. AntiSMASH analysis identified three siderophores with high similarity, and improved MALDI-TOF IMS confirmed the presence of the siderophore pyochelin. Furthermore, the BX1 filtrate suppressed the expression of virulence genes in C. fructicola and induced the expression of disease resistance genes in banana. However, the presence of 80 µM iron ions notably mitigated BX1's inhibitory effects and reversed the changes in related gene expression. These results underscore BX1's robust efficacy as a biocontrol agent in managing banana anthracnose, highlight the effective antifungal compounds, and elucidate the influence of environmental factors on biocontrol effectiveness.

Response and disease resistance evaluation of sorghum seedlings under anthracnose stress.

Sorghum is the world's fifth-largest cereal crop, and anthracnose (Colletotrichum sublineola) is the main disease affecting sorghum. However, systematic research on the cellular structure, physiological and biochemical, and genes related to anthracnose resistance and disease resistance evaluation in sorghum is lacking in the field. Upon inoculation with anthracnose (C. sublineola) spores, disease-resistant sorghum (gz93) developed a relative lesion area (RLA) that was significantly smaller than that of the disease-susceptible sorghum (gz234). The leaf thickness, length and profile area of leaf mesophyll cells, upper and lower epidermal cells decreased in the lesion area, with a greater reduction observed in gz234 than in gz93. The damage caused by C. sublineola resulted in a greater decrease in the net photosynthetic rate (Pn) in gz234 than in gz93, with early-stage reduction due to stomatal limitation and late-stage reduction caused by lesions. Overall, the activities of superoxide dismutase (SOD) and catalase (CAT), the content of proline (Pro), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and gibberellic acid (GA3), are higher in gz93 than in gz234 and may be positively correlated with disease resistance. While malondialdehyde (MDA) may be negatively correlated with disease resistance. Disease-resistant genes are significantly overexpressed in gz93, with significant expression changes in gz234, which is related to disease resistance in sorghum. Correlation analysis indicates that GA3, MDA, peroxidase (POD), and disease-resistance genes can serve as reference indicators for disease severity. The regression equation RLA = 0.029 + 8.02 × 10-6 JA-0.016 GA3 can predict and explain RLA. Principal component analysis (PCA), with the top 5 principal components for physiological and biochemical indicators and the top 2 principal components for disease-resistant genes, can explain 82.37% and 89.11% of their total variance, reducing the number of evaluation indicators. This study provides a basis for research on the mechanisms and breeding of sorghum with resistance to anthracnose.

Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples.

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/µL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.