Haplotypes at the sorghum ARG4 and ARG5 NLR loci confer resistance to anthracnose.

Sorghum anthracnose caused by the fungus Colletotrichum sublineola (Cs) is a damaging disease of the crop. Here, we describe the identification of ANTHRACNOSE RESISTANCE GENES (ARG4 and ARG5) encoding canonical nucleotide-binding leucine-rich repeat (NLR) receptors. ARG4 and ARG5 are dominant resistance genes identified in the sorghum lines SAP135 and P9830, respectively, that show broad-spectrum resistance to Cs. Independent genetic studies using populations generated by crossing SAP135 and P9830 with TAM428, fine mapping using molecular markers, comparative genomics and gene expression studies determined that ARG4 and ARG5 are resistance genes against Cs strains. Interestingly, ARG4 and ARG5 are both located within clusters of duplicate NLR genes at linked loci separated by ~1 Mb genomic region. SAP135 and P9830 each carry only one of the ARG genes while having the recessive allele at the second locus. Only two copies of the ARG5 candidate genes were present in the resistant P9830 line while five non-functional copies were identified in the susceptible line. The resistant parents and their recombinant inbred lines carrying either ARG4 or ARG5 are resistant to strains Csgl1 and Csgrg suggesting that these genes have overlapping specificities. The role of ARG4 and ARG5 in resistance was validated through sorghum lines carrying independent recessive alleles that show increased susceptibility. ARG4 and ARG5 are located within complex loci displaying interesting haplotype structures and copy number variation that may have resulted from duplication. Overall, the identification of anthracnose resistance genes with unique haplotype stucture provides a foundation for genetic studies and resistance breeding.

ANTHRACNOSE RESISTANCE GENE2 confers fungal resistance in sorghum.

Sorghum is an important food and feed crop globally; its production is hampered by anthracnose disease caused by the fungal pathogen Colletotrichum sublineola (Cs). Here, we report identification and characterization of ANTHRACNOSE RESISTANCE GENE 2 (ARG2) encoding a nucleotide-binding leucine-rich repeat (NLR) protein that confers race-specific resistance to Cs strains. ARG2 is one of a cluster of several NLR genes initially identified in the sorghum differential line SC328C that is resistant to some Cs strains. This cluster shows structural and copy number variations in different sorghum genotypes. Different sorghum lines carrying independent ARG2 alleles provided the genetic validation for the identity of the ARG2 gene. ARG2 expression is induced by Cs, and chitin induces ARG2 expression in resistant but not in susceptible lines. ARG2-mediated resistance is accompanied by higher expression of defense and secondary metabolite genes at early stages of infection, and anthocyanin and zeatin metabolisms are upregulated in resistant plants. Interestingly, ARG2 localizes to the plasma membrane when transiently expressed in Nicotiana benthamiana. Importantly, ARG2 plants produced higher shoot dry matter than near-isogenic lines carrying the susceptible allele suggesting an absence of an ARG2 associated growth trade-off. Furthermore, ARG2-mediated resistance is stable at a wide range of temperatures. Our observations open avenues for resistance breeding and for dissecting mechanisms of resistance.

A Colletotrichum tabacum Effector Cte1 Targets and Stabilizes NbCPR1 to Suppress Plant Immunity.

Colletotrichum tabacum, causing anthracnose in tobacco, is a notorious plant pathogen threatening tobacco production globally. The underlying mechanisms of C. tabacum effectors that interfere with plant defense are not well known. Here, we identified a novel effector, Cte1, from C. tabacum, and its expression was upregulated in the biotrophic stage. We found that Cte1 depresses plant cell death initiated by BAX and inhibits reactive oxygen species (ROS) bursts triggered by flg22 and chitin in Nicotiana benthamiana. The CTE1 knockout mutants decrease the virulence of C. tabacum to N. benthamiana, and the Cte1 transgenic N. benthamiana increase susceptibility to C. tabacum, verifying that Cte1 is involved in the pathogenicity of C. tabacum. We demonstrated that Cte1 interacted with NbCPR1, a Constitutive expresser of Plant Resistance (CPR) protein in plants. Silencing of NbCPR1 expression attenuated the infection of C. tabacum, indicating that NbCPR1 negatively regulates plant immune responses. Cte1 stabilizes NbCPR1 in N. benthamiana. Our study shows that Cte1 suppresses plant immunity to facilitate C. tabacum infection by intervening in the native function of NbCPR1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Global transcriptome analysis reveals resistance genes in the early response of common bean (Phaseolus vulgaris L.) to Colletotrichum lindemuthianum.

BACKGROUND: Disease can drastically impair common bean (Phaseolus vulgaris L.) production. Anthracnose, caused by the fungal pathogen Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara, is one of the diseases that are widespread and cause serious economic loss in common bean. RESULTS: Transcriptome analysis of the early response of common bean to anthracnose was performed using two resistant genotypes, Hongyundou and Honghuayundou, and one susceptible genotype, Jingdou. A total of 9,825 differentially expressed genes (DEGs) responding to pathogen infection and anthracnose resistance were identified by differential expression analysis. By using weighted gene coexpression network analysis (WGCNA), 2,051 DEGs were found to be associated with two resistance-related modules. Among them, 463 DEGs related to anthracnose resistance were considered resistance-related candidate genes. Nineteen candidate genes were coexpressed with three resistance genes, Phvul.001G243600, Phvul.001G243700 and Phvul.001G243800. To further identify resistance genes, 46 candidate genes were selected for experimental validation using salicylic acid (SA) and methyl jasmonate (MeJA). The results indicated that 38 candidate genes that responded to SA/MeJA treatment may be involved in anthracnose resistance in common bean. CONCLUSIONS: This study identified 38 resistance-related candidate genes involved in the early response of common bean, and 19 resistance-related candidate genes were coexpressed with anthracnose resistance genes. This study identified putative resistance genes for further resistance genetic investigation and provides an important reference for anthracnose resistance breeding in common bean.

Exploring the genetic basis of anthracnose resistance in Ethiopian sorghum through a genome-wide association study.

BACKGROUND: Sorghum anthracnose is a major disease that hampers the productivity of the crop globally. The disease is caused by the hemibiotrophic fungal pathogen Colletotrichum sublineola. The identification of anthracnose-resistant sorghum genotypes, defining resistance loci and the underlying genes, and their introgression into adapted cultivars are crucial for enhancing productivity. In this study, we conducted field experiments on 358 diverse accessions of Ethiopian sorghum. Quantitative resistance to anthracnose was evaluated at locations characterized by a heavy natural infestation that is suitable for disease resistance screening. RESULTS: The field-based screening identified 53 accessions that were resistant across locations, while 213 accessions exhibited variable resistance against local pathotypes. Genome-wide association analysis (GWAS) was performed using disease response scores on 329 accessions and 83,861 single nucleotide polymorphisms (SNPs) generated through genotyping-by-sequencing (GBS). We identified 38 loci significantly associated with anthracnose resistance. Interestingly, a subset of these loci harbor genes encoding receptor-like kinases (RLK), nucleotide-binding leucine-rich repeats (NLRs), stress-induced antifungal tyrosine kinase that have been previously implicated in disease resistance. A SNP on chromosome 4 (S04_66140995) and two SNPs on chromosome 2 (S02_75784037, S02_2031925), localized with-in the coding region of genes that encode a putative stress-induced antifungal kinase, an F-Box protein, and Xa21-binding RLK that were strongly associated with anthracnose resistance. We also identified highly significant associations between anthracnose resistance and three SNPs linked to genes (Sobic.002G058400, Sobic.008G156600, Sobic.005G033400) encoding an orthologue of the widely known NLR protein (RPM1), Leucine Rich Repeat family protein, and Heavy Metal Associated domain-containing protein, respectively. Other SNPs linked to predicted immune response genes were also significantly associated with anthracnose resistance. CONCLUSIONS: The sorghum germplasm collections used in the present study are genetically diverse. They harbor potentially useful, yet undiscovered, alleles for anthracnose resistance. This is supported by the identification of novel loci that are enriched for disease resistance regulators such as NLRs, LRKs, Xa21-binding LRK, and antifungal proteins. The genotypic data available for these accessions offer a valuable resource for sorghum breeders to effectively improve the crop. The genomic regions and candidate genes identified can be used to design markers for molecular breeding of sorghum diseases resistance.

Genome-wide identification analysis of the 4-Coumarate: CoA ligase (4CL) gene family expression profiles in Juglans regia and its wild relatives J. Mandshurica resistance and salt stress.

Persian walnut (Juglans regia) and Manchurian walnut (Juglans mandshurica) belong to Juglandaceae, which are vulnerable, temperate deciduous perennial trees with high economical, ecological, and industrial values. 4-Coumarate: CoA ligase (4CL) plays an essential function in plant development, growth, and stress. Walnut production is challenged by diverse stresses, such as salinity, drought, and diseases. However, the characteristics and expression levels of 4CL gene family in Juglans species resistance and under salt stress are unknown. Here, we identified 36 Jr4CL genes and 31 Jm4CL genes, respectively. Based on phylogenetic relationship analysis, all 4CL genes were divided into three branches. WGD was the major duplication mode for 4CLs in two Juglans species. The phylogenic and collinearity analyses showed that the 4CLs were relatively conserved during evolution, but the gene structures varied widely. 4CLs promoter region contained multiply cis-acting elements related to phytohormones and stress responses. We found that Jr4CLs may be participated in the regulation of resistance to anthracnose. The expression level and some physiological of 4CLs were changed significantly after salt treatment. According to qRT-PCR results, positive regulation was found to be the main mode of regulation of 4CL genes after salt stress. Overall, J. mandshurica outperformed J. regia. Therefore, J. mandshurica can be used as a walnut rootstock to improve salt tolerance. Our results provide new understanding the potential functions of 4CL genes in stress tolerance, offer the theoretical genetic basis of walnut varieties adapted to salt stress, and provide an important reference for breeding cultivated walnuts for stress tolerance.

Emerging roles of plant microRNAs during Colletotrichum spp. infection.

MAIN CONCLUSION: This review provides valuable insights into plant molecular regulatory mechanisms during fungus attacks, highlighting potential miRNA candidates for future disease management. Plant defense responses to biotic stress involve intricate regulatory mechanisms, including post-transcriptional regulation of genes mediated by microRNAs (miRNAs). These small RNAs play a vital role in the plant's innate immune system, defending against viral, bacterial, and fungal attacks. Among the plant pathogenic fungi, Colletotrichum spp. are notorious for causing anthracnose, a devastating disease affecting economically important crops worldwide. Understanding the molecular machinery underlying the plant immune response to Colletotrichum spp. is crucial for developing tools to reduce production losses. In this comprehensive review, we examine the current understanding of miRNAs associated with plant defense against Colletotrichum spp. We summarize the modulation patterns of miRNAs and their respective target genes. Depending on the function of their targets, miRNAs can either contribute to host resistance or susceptibility. We explore the multifaceted roles of miRNAs during Colletotrichum infection, including their involvement in R-gene-dependent immune system responses, hormone-dependent defense mechanisms, secondary metabolic pathways, methylation regulation, and biosynthesis of other classes of small RNAs. Furthermore, we employ an integrative approach to correlate the identified miRNAs with various strategies and distinct phases of fungal infection. This study provides valuable insights into the current understanding of plant miRNAs and their regulatory mechanisms during fungus attacks.

Biological characterization of emerging fungal pathogen Colletotrichum associated with mango (Mangifera indica L.) post-harvest anthracnose from Vietnam.

BACKGROUND: Post-harvest anthracnose (PHA) of mango is a devastating disease, which results in huge loss to mango producers and importers. Various species of PHA, diverse pathogenicity, and different resistance towards fungicides make it essential to evaluate the pathogen taxonomic status and biological characterization. METHODS AND RESULTS: Two strains DM-1 and DM-2 isolated from the fruit of DaQing mango from Vietnam were identified as Colletotrichum fructicola and C. asianum respectively, based on the morphological features, along with the phylogenetic tree of ITS and ApMat combined sequences. The growth status of different Colletotrichum strains under different conditions was analyzed to reveal the biological characteristics. The optimum growth temperature of DM-1 and DM-2 was 28 °C and mycelia grew rapidly in the dark. Both strains could grow in media with pH 4-11, while the optimum pH value was 6. Maltose and soluble starch were the most suitable carbon source for DM-1 and DM-2 respectively, and the peptone was the most suitable nitrogen source for both strains. The lethal temperatures were recorded as 55 °C 5 min for DM-1, and 50 °C 10 min for DM-2. CONCLUSIONS: To the best of our knowledge, it is the first study reporting the identification of the pathogens: C. fructicola and C. asianum responsible for postharvest fruit anthracnose of mango in Vietnam.

Mapping of adult plant recessive resistance to anthracnose in Indian common bean landrace Baspa/KRC 8.

BACKGROUND: The common bean (Phaseolus vulgaris) has become the food of choice owing to its wealthy nutritional profile, leading to a considerable increase in its cultivation worldwide. However, anthracnose has been a major impediment to production and productivity, as elite bean cultivars are vulnerable to this disease. To overcome barriers in crop production, scientists worldwide are working towards enhancing the genetic diversity of crops. One way to achieve this is by introducing novel genes from related crops, including landraces like KRC 8. This particular landrace, found in the North Western Himalayan region, has shown adult plant resistance against anthracnose and also possesses a recessive resistance gene. METHODS AND RESULTS: In this study, a population of 179 F2:9 RIL individuals (Jawala × KRC 8) was evaluated at both phenotypic and genotypic levels using over 830 diverse molecular markers to map the resistance gene present in KRC 8. We have successfully mapped a resistance gene to chromosome Pv01 using four SSR markers, namely IAC 238, IAC 235, IAC 259, and BM 146. The marker IAC 238 is closely linked to the gene with a distance of 0.29 cM, while the other markers flank the recessive resistance gene at 10.87 cM (IAC 259), 17.80 cM (BM 146), and 25.22 cM (IAC 235). Previously, a single recessive anthracnose resistance gene (co-8) has been reported in the common bean accession AB 136. However, when we performed PCR amplification with our tightly linked marker IAC 238, we got different amplicons in AB 136 and KRC 8. Interestingly, the susceptible cultivar Jawala produced the same amplicon as AB 136. This observation indicated that the recessive gene present in KRC 8 is different from co-8. As the gene is located far away from the Co-1 locus, we suggest naming the recessive gene co-Indb/co-19. Fine mapping of co-Indb in KRC 8 may provide new insights into the cloning and characterization of this recessive gene so that it can be incorporated into future bean improvement programs. Further, the tightly linked marker IAC 238 can be utilized in marker assisted introgression in future bean breeding programs. CONCLUSION: The novel co-Indb gene present in Himalayan landrace KRC 8, showing adult plant resistance against common bean anthracnose, is independent from all the resistance genes previously located on chromosome Pv01.

Suppression of anthracnose disease by orsellinaldehyde isolated from the mushroom Coprinus comatus.

AIMS: Anthracnose caused by Colletotrichum species is one of the most devastating diseases of fruits and crops. We isolated and identified an antifungal compound from the mushroom Coprinus comatus and investigated its inhibitory potential against anthracnose disease-causing fungi with the goal of discovering natural products that can suppress anthracnose-caused plant disease. METHODS AND RESULTS: The culture filtrate of C. comatus was subjected to a bioassay-guided isolation of antifungal compounds. The active compound was identified as orsellinaldehyde (2,4-dihydroxy-6-methylbenzaldehyde) based on mass spectroscopy and nuclear magnetic resonance analyses. Orsellinaldehyde displayed broad-spectrum inhibitory activity against different plant pathogenic fungi. Among the tested Colletotrichum species, it exhibited the lowest IC50 values on conidial germination and germ tube elongation of Colletotrichum orbiculare. The compound also showed remarkable inhibitory activity against Colletotrichum gloeosporiodes. The staining of Colletotrichum conidia with fluorescein diacetate and propidium iodide demonstrated that the compound is fungicidal. The postharvest in-vivo detached fruit assay indicated that orsellinaldehyde suppressed anthracnose lesion symptoms on mango and cucumber fruits caused by C. gloeosporioides and C. orbiculare, respectively. CONCLUSIONS: Orsellinaldehyde was identified as a potent antifungal compound from the culture filtrate of C. comatus. The inhibitory and fungicidal activities of orsellinaldehyde against different Colletotrichum species indicate its potential as a fungicide for protecting various fruits against anthracnose disease-causing fungi.

Antifungal efficacy of biogenic waste derived colloidal/nanobiochar against Colletotrichum gloeosporioides species complex.

Anthracnose caused by Colletotrichum spp. usually resulting in significant postharvest losses in the banana production chain. This study investigated the inhibitory effect of corn cob colloidal/nanobiochar (CCN) and Gliricidia sepium wood colloidal/nanobiochar (GCN) on the Colletotrichum gloeosporioides species complex. The CCN and GCN materials were synthesized and thoroughly characterized using various techniques, including UV-Vis and Fluorescence spectroscopy. Then after the fungal growth was examined on Potato Dextrose Agar (PDA) media supplemented with different CCN and GCN concentrations of 0.4 - 20 g/L and CCN and GCN with zeolite at various weight percentages of 10% to 50% w/w. Results from the characterization revealed that CCN exhibited a strong UV absorbance peak value of 0.630 at 203 nm, while GCN had a value of 0.305 at 204 nm. In terms of fluorescence emission, CCN displayed a strong peak intensity of 16,371 at 412 nm, whereas GCN exhibited a strong peak intensity of 32,691 at 411 nm. Both CCN and GCN, at concentrations ranging from 1 to 8 and 0.4 - 20 g/L, respectively, displayed notable reductions in mycelial densities and inhibited fungal growth compared to the control. Zeolite incorporation further enhanced the antifungal effect. To the best of our knowledge, this is the first study to demonstrate the promising potential of colloidal/nanobiochar in effectively controlling anthracnose disease. The synthesized CCN and GCN demonstrate promising antifungal potential against Colletotrichum gloeosporioides species complex, offering the potential for the development of novel and effective antifungal strategies for controlling anthracnose disease in Musa spp.

A Sorghum F-Box Protein Induces an Oxidative Burst in the Defense Against Colletotrichum sublineola.

The hemibiotrophic fungal pathogen Colletotrichum sublineola is the causal agent of anthracnose in sorghum (Sorghum bicolor), resulting in leaf blight, stalk rot, and head blight in susceptible genotypes, with yield losses of up to 50%. The development of anthracnose-resistant cultivars can reduce reliance on fungicides and provide a more sustainable and economical means for disease management. A previous genome-wide association study of the sorghum association panel identified the candidate resistance gene Sobic.005G172300 encoding an F-box protein. To better understand the role of this gene in the defense against C. sublineola, gene expression following infection with C. sublineola was monitored by RNA sequencing in seedlings of sorghum accession SC110, which harbored the resistance allele, and three accessions that harbored a susceptible allele. Only in SC110 did the expression of Sobic.005G172300 increase during the biotrophic phase of infection. Subsequent transcriptome analysis, gene co-expression networks, and gene regulatory networks of inoculated and mock-inoculated seedlings of resistant and susceptible accessions suggest that the increase in expression of Sobic.005G172300 induces an oxidative burst by lowering the concentration of ascorbic acid during the biotrophic phase of infection. Based on gene regulatory network analysis, the protein encoded by Sobic.005G172300 is proposed to target proteins involved in the biosynthesis of ascorbic acid for polyubiquitination through the SCF E3 ubiquitin ligase, causing their degradation via the proteasome.

First report of anthracnose caused by Colletotrichum grevilleae on apple in Korea.

In October 2022, typical symptoms of anthracnose were observed on apple (Malus ⅹ domestica cv. Fuji) fruits collected from Pocheon in Gyeonggi province, South Korea (N37.98074°, E127.33995°). In the surveyed orchard, the incidence rate of apple anthracnose was less than 1%. The initial symptoms were brown-to-dark brown lesions, and with disease progression, they enlarged and the pulp became soft, forming a brown band. In total 29 apple fruits were collected, and the causal agent was isolated by removing the peel, and the diseased tissues were directly transferred onto potato dextrose agar (PDA), followed by incubation for 7 days at 25°C. As the results, two isolates (GgPc22-1-11 and GgPc22-1-13) were obtained. For describing morphological and cultural characteristics, isolate GgPc22-1-11 was cultured on PDA and synthetic nutrient-poor agar (SNA) at 25°C under near-UV light with a 12-h photoperiod for 10 days. The colonies of GgPc22-1-11 on PDA were initially white and subsequently appeared light gray to olivaceous with white margins. The reverse side of the plates were dark brown and slate blue (Supplementary Fig. S1). Colonies on SNA were flat with an entire margin and short sparse white aerial mycelium. No setae were observed. Conidia on PDA were hyaline, straight, aseptate with a rounded apex, clavate to cylindrical, and measured 16.4 ± 2.4 (10.8-23.8) × 5.5 ± 0.7 (3.6-7.7) µm (n = 200). Appressoria were medium-to-dark brown, aseptate, solitary or in groups with irregular outlines, and lobate or having undulate margins (Supplementary Fig. S1). These morphological and cultural characteristics of GgPc22-1-11 were consistent with those of Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, pathogens of Proteaceae and Punica granatum (Liu et al. 2013; Huang et al. 2023). DNA was extracted from GgPc22-1-11, PCR was performed and Phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of rDNA, ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT) genes was conducted (Weir et al. 2012). The resulting sequences were deposited in GenBank under the accession numbers LC773710-LC773714. A nucleotide BLAST search revealed that the ITS sequences of the isolates were 98.95% identical to those of C. grossum CAUG7 (KP890165.1). The TUB2, GAPDH, CHS-1, and ACT sequences of the isolates were 99.79%, 99.24%, 100%, and 100%, respectively, identical to those of C. grevilleae WP4. GgPc22-1-11 was clustered with C. grevilleae WP4 using neighbor joining analysis conducted with MEGA X software (Kumar et al. 2018) (Supplementary Fig. S2). Pathogenicity tests were conducted using GgPc22-1-11 and repeated three times. A total of 12 symptomless apples of each variety were selected, including Fuji, Hongro, Tsugaru, and RubyS. The apples were surface-sterilized with 70% ethanol and wounded using a sterile needle. Both wounded and unwounded apples were inoculated with mycelium plugs and paper disks containing a conidial suspension (1 × 106 conidia/ml) and placed in a plastic box with moist paper towels (>90% relative humidity) at 25°C in dark. At 5 days after inoculation, all artificially wounded fruits exhibited symptoms and 30% (4 out of 12) of unwounded inoculated fruits showed symptoms in each apple variety while control fruits were asymptomatic both the unwounded and wounded inoculations (Supplementary Fig. S1). To fulfill Koch's postulates, the fungi were reisolated from symptomatic tissues and were identical to GgPc22-1-11 confirmed by morphological and molecular analysis. To the best of our knowledge, C. grevilleae has been reported in Protea sp. and pomegranate (Liu et al. 2013; Huang et al. 2023) but not in apples to date, and this is the first report of C. grevilleae causing anthracnose in apple fruits. This research of the newly emerged unreported Colletotrichum species can offer valuable information for development of an effective fungicide spray program to control apple anthracnose.

Race Structure and Molecular Diversity of Colletotrichum lindemuthianum of Common Bean in Zambia.

Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is a major disease of common bean (Phaseolus vulgaris L.) worldwide. C. lindemuthianum is genetically highly variable, and understanding the pathogen's diversity and distribution is a key step in developing common bean varieties with durable anthracnose resistance. The objectives of this study were to (i) characterize the race structure of C. lindemuthianum in Zambia and (ii) assess the molecular diversity of C. lindemuthianum in Zambia. A field survey was conducted in 20 bean-growing districts in Zambia to collect anthracnose symptomatic bean plants. A total of 103 C. lindemuthianum isolates were collected and characterized based on their reactions on 12 common bean race differential cultivars. RAM and ERIC-BOX DNA markers were used to assess molecular diversity of 60 isolates. A total of 58 races were characterized from the 103 isolates. Race 5 was the least virulent, and race 1631 was the most virulent based on their reaction on the 12 race differential cultivars. Race 19 had the highest recovery frequency (11%) and was the most extensively dispersed among the 22 bean-growing districts from where the isolates were collected. Only six races had previously been reported in Zambia, and 52 races were identified as new races reported for the first time in Zambia. Two races were virulent only on Andean cultivars, 11 races were virulent only on Middle American cultivars, and 45 races were virulent on both Andean and Middle American cultivars. No individual isolate showed pathogenicity on all the differential cultivars, and no isolate overcame the Co-4, Co-5, and Co-7 resistance gene pyramid that naturally exists in G2333. Phylogenetic analysis categorized the 60 isolates in six major clusters and six subclusters. The 60 isolates showed high genetic heterogeneity among and within a race of the same virulence. The study has revealed the existence of both Andean and Middle American races and extensive molecular diversity of C. lindemuthianum in Zambia. The knowledge on the race structure of C. lindemuthianum that this study has provided will be valuable for making breeding decisions on the host plant resistance genes required for developing common bean varieties with durable resistance to anthracnose in Zambia.

First Report of Colletotrichum fioriniae Causing Anthracnose on Persimmon in China.

Persimmons (Diospyros kaki Thunb.) have a longstanding history of cultivation in China. Both aesthetically pleasing and edible, they often symbolize a sweet and fulfilling life. During the summer of 2022, a severe outbreak of anthracnose was observed on the lower leaves of persimmon trees in the National Field Genebank for Persimmon (NFGP), located in Yangling, Shaanxi, China (34°17'42.80″ N, 108°04'08.21″ E). The estimated incidence rate of this disease within the NFGP was approximately 30%. The typical symptoms of the disease included the presence of irregular lesions on leaves, and oval sunken lesions on infected fruit. Under high humidity conditions, pink sticky substances appeared in the affected areas. The presence of numerous lesions led to softening and detachment of persimmon fruit. To identify the causal pathogen, 5 × 5mm samples of the diseased leaves were collected from the interface between the infected and healthy leaves. The leaves were disinfected with 70% alcohol for 20 s, followed by rinsing with sterile water. Subsequently, the leaves were immersed in 1% NaClO for 2 to 3 minutes, rinsed with sterile water three times, dried using sterile absorbent paper, and the leaf samples were then transferred onto potato dextrose agar (PDA) medium, and cultured in 25°C incubators. Once the colony reached a certain size, small pieces of hyphae were extracted from edge and transferred for purification and repeated three times. After being cultured on PDA for 7 days, the colony showed a white spongy surface with a pink-orange center. The conidia displayed a fusiform shape and were transparent, measuring 4.58 to 6.53 µm × 9.27 to 13.11 µm (n=50). The isolates share morphological similarities with Colletotrichum fioriniae. The representative isolate HY-7 was selected for molecular identification. The internal transcribed spacers (ITS) region, chitin synthase (CHS-1), actin (ACT), beta-tubulin 2 (TUB2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were amplified using ITS1/4 (White et al. 1990), CHS-79F/CHS-345R (Carbone & Kohn, 1999), ACT512F/ACT (Carbone & Kohn, 1999), T1/BT2B (Glass & Donaldson 1995, O'Donnell et al., 1997), and GDF/GDR (Templeton et al. 1992), respectively. The generated sequences were deposited at GenBank under accession numbers OR878056 (ITS), OR766019 (CHS-1), OR766021(TUB2), OR766018 (ACT) and OR766020 (GAPDH). BLAST analysis revealed the sequences were 100% identical to C. fioriniae (MH865005 for ITS, JQ948953 for CHS-1, JQ949613 for ACT, JQ949943 for TUB2 and JQ948622 for GAPDH). The morphological characteristics and molecular analyses of the isolate matched the description of C. fioriniae. To fulfill Koch's postulates, the twigs and leaves of 'Fupingjianshi' in four different directions were inoculated without wounding in the field, and 10 healthy fruits were selected for wound inoculation. The concentration of conidia used for inoculation was about 1 × 106 conidia/ml, and sterilized water was used as control. The experiment was replicated three times under the same conditions. One week after inoculation, characteristic symptoms resembling those observed on the leaves of primary diseased persimmon trees appeared on the leaves and fruits. No symptoms were observed on the leaves, twigs and fruits in the control treatment. The pathogen from the artificially infected leaves and fruits were reisolated and identified as C. fiorinae based on morphological and molecular characteristics. Persimmon anthracnose is a common disease in regions where the fruit is grown, to the best of our knowledge, this is the first documented occurrence of C. fioriniae-induced anthracnose on persimmons in China, which should be paid more attentions. This report will help identify disease symptoms in the field and provides a basis for determining the occurrence, distribution, and control of C. fioriniae on persimmon leaves and fruits.

First Report of Colletotrichum fructicola Causing Anthracnose on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim., belonging to the family Berberidaceae and genus Epimedium, is a perennial herb widely studied for its anti-osteoporosis, anti-cancer, and anti-sexual-dysfunction effects in Asian countries (Tan et al. 2016; Zhang et al. 2016). High levels of bioactive chemicals in Epimedium spp. has endowed it with important clinical and commercial values (Liu et al. 2013). In September 2021, a leaf disease was found in Zhumadian City, China (32°58'12" N, 114°37'48" E). Survey statistics indicated that disease prevalence in a 266-ha planting area was approximately 29.6%. The lesions appeared at the leaf tips, gradually enlarged, and were brown with a yellow halo. Further, the lesions were dry with distributed black spots. Thirty infected leaves collected from five sites within the planting base . The collected leaves were cut into 5×5 mm pieces , surface-sterilized in 75% alcohol for 15 s, triple washed with sterile ddH2O, disinfested with 0.1% HgCl2 solution for 30 s (Liu et al. 2021), triple washed again with sterilized ddH2O, and then placed onto PDA and incubated in the dark for 3 d at 28°C. Subsequently, five fungal strains were purified; among them, only the isolate HY3-2 infected the host plant and was selected for further morphological characterization. The colonies of HY3-2 initially appeared white, their mycelia became gray at the center after 4 d, and orange-red conidial clumps appeared in them after 7 d. Conidia (10.0-19.5 µm × 4.5-5.6 µm, n=50) were single celled, nearly spherical or stick-shaped and colorless. Morphological characteristics of the isolate were consistent with those of Colletotrichum species. Additionally, glycerol-3-phosphate dehydrogenase (gapdh), actin (act), calmodulin (cal), ß-tubulin 2 (tub2), and chitin synthase-1 (chs-1), (Weir et al. 2012) were amplified and sequenced using the primers GDF/GDR, ACT-512F/783R, CL1C/CL2C, T1/Bt2b, and CHS-79F/354R, respectively for molecular identification. The resulting sequences were deposited in GenBank: gapdh (ON351609), act (ON351608), tub2 (ON351610), chs-1 (ON532788), and cal (ON532787). Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method (Zhang et al. 2020). The phylogenetic tree showed that the isolate belongs to C. fructicola clade with a credibility value of 85%.To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the isolate HY3-2 were prepared with sterile ddH2O to infect the leaves. Ninety healthy leaves from 30 plants in pots were punctured using a sterilized needle (Huang et al. 2022), and inoculated by spraying the conidial suspension on the leaves in a greenhouse at 25°C and 80% relative humidity. In the control plants, the suspension was replaced with water. After 7 d, the inoculated plants showed symptoms similar to those of the original infected plant, whereas the control showed no symptoms. C. fructicola was isolated and identified again as previously described. A pathogenicity test was also conducted in the field using the same method as that used in the greenhouse in July 2022, the results of which were consistent with those of the greenhouse. In China, C. fructicola has been reported on Walnut (Wang et al. 2022), Punica granatum (Hu et al. 2023) and others. To our knowledge, this is the first report of C. fructicola causing anthracnose in E. sagittatum in China. This report provides an important basis for further disease control research.

First Report of Anthracnose Caused by Colletotrichum spaethianum on Zephyranthes candida in China.

Zephyranthes candida, an bulbous perennial plant, are planted in almost every park. In October 2023, anthracnose symptoms were observed on Z. candida leaves in Jiangxi Agricultural University (28.75° N, 115.83°E), Nanchang, Jiangxi Province, China, and the incidence of disease were up to 35% (140 of 400 plants). The lesions extended from the leaf apex to the base, appearing as a dark brown color, and later changed to yellow and became dry. To isolate the pathogen, 20 symptomatic leaves were collected and cut into small pieces (4×4 mm, one pieces per leave), surface-sterilized with 70% ethanol for 10 s and 1% NaClO for 30 s, rinsed thrice with sterile water, placed onto potato dextrose agar (PDA) plates and incubated at 25℃ for 5 days. Fifteen isolates (15 out of 20) with similar morphological characteristics were obtained. The colonies on PDA presented effuse mycelium, initially white and later pale gray. Conidia were hyaline, curved or slightly curved, aseptate, with a truncate base and acute apex, measuring 17 to 23 × 3 to 6 µm (n = 50), and were matched to Colletotrichum species (Damm et al. 2009). To further confirm species, two representative isolates (JFRL 03-2873 and JFRL 03-2874) were selected for molecular identification. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), histone 3 (HIS3), actin (ACT) and ß-tubulin 2 (TUB2) regions were amplified and sequenced by using primers sets ITS5/ITS4, Gpd1/Gpd2, CHS-79F/CHS354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R and T1/Bt2b (Tan et al. 2022), respectively. These sequences were deposited into GenBank with accession number PP425890-PP425891 (ITS), PP437551-PP437552 (GAPDH), PP437549-PP437550 (CHS), PP480643-PP480644 (HIS3), PP437547-PP437548 (ACT) and PP437553-PP437554 (TUB2). A BLASTN search revealed high similarity of 99%-100% to ITS (GU227807, 518 nt/519 nt), GAPDH (GU228199, 525 nt/526 nt), CHS (GU228297, 251 nt/251 nt), HIS3 (GU228003, 372 nt/373 nt), ACT (GU227905, 236 nt/237 nt) and TUB2 (GU228101, 490 nt/490 nt) sequences of Colletotrichum spaethianum CBS 167.49. A maximum likelihood phylogenetic tree was constructed by combining ITS, GAPDH, CHS , HIS3, ACT and TUB2 sequences in IQtree web server (Ngugen et al. 2015). The result indicated that the two representative isolates were clustered together with Colletotrichum spaethianum in a clade with 100% bootstrap support. Based on morphological observation and sequence analysis, the isolates were identified as C. spaethianum. To confirm pathogenicity, six surface-sterilized leaves of Z. candida were wounded and inoculated with 1 × 106 conidia/ml conidial suspension of JFRL 03-2873, and control leaves were inoculated with sterile water. They were incubated at 25 ℃ with 12 h photoperiod and 80% humidity, the experiment was repeated twice. After five days, all leaves inoculated with JFRL 03-2873 displayed anthracnose symptom, whereas the control leaves remained unaffected. We re-isolated C. spaethianum from the symptomatic leaves and identified it based on morphological and molecular characteristics. Previous studies reported that C. spaethianum caused anthracnose on various common herbaceous plants in China (Vieira et al. 2014, Guo et al. 2013), but to our knowledge, this is the first report of C. spaethianum causing anthracnose on Z. candida in China. Anthracnose disease caused great economic loss to the cultivation of landscape plant Z. candida. Therefore, it is necessary to pay more attention to the anthracnose disease of herbaceous plants caused by C. spaethianum and develop appropriate control strategies.

Genomic Insights into Combating Anthracnose with an Endophytic Bacillus amyloliquefaciens Strain.

Anthracnose, caused by Colletotrichum spp., is a common disease of Camellia oleifera. In this study, a Bacillus amyloliquefaciens strain, GZY63, was isolated from fruit of the anthracnose-resistant cultivar of Ca. oleifera "Ganzhouyou7." Plate confrontation assays and field experiments demonstrated the strong inhibitory effect of GZY63 on anthracnose, and this strain exhibited broad-spectrum resistance to nine pathogenic Colletotrichum spp. This strain shows potential as a fungicide alternative, but genetic information on this strain is critical for its optimal use. Combining Illumina and Nanopore sequencing, we assembled a high-quality circular genome of GZY63 that contained no plasmids. The GZY63 complete genome was approximately 3.93 Mb and had an average guanine-cytosine content of 46.5%. The genome comprised 4,024 predicted coding sequences and 12 types of gene clusters involved in secondary metabolite production. This genome information provides insights into the mechanism underlying the antagonistic impact of the GZY63 strain on anthracnose and its symbiotic relationship with Ca. oleifera.

First Report of Yellow Leaf Spots on Maize Caused by Colletotrichum siamense in Yunnan Province, China.

The southwest maize planting area is the third largest maize-producing region in China, including the entire provinces of Sichuan, Yunnan and Guizhou, parts of Guangxi and Hunan provinces. In June 2022, yellow leaf spot symptoms were observed commonly on maize in southern Yunnan province, including Pu'er City, Xishuangbanna Dai autonomous prefecture and Honghe Hani & Yi autonomous prefecture. The disease incidence on maize in Pu'er ranged from 10% to 20% from June to August. The initial symptoms appeared as needle-like spots scattered on the leaf surface with obvious yellow haloes, with a diameter ranging from 0.2 to 2 mm and were quite similar to maize Curvularia leaf spot. But the lesion size did not expand significantly and without reddish or dark brown margins. In July 2023, 30 diseased leaves were collected in Pu'er City, Yunnan Province. Leaf tissues (3×3 mm) were cut from the infected margins, surface disinfested with 75% ethanol for 30 s, 2% sodium hypochlorite for 2 min, and rinsed three times with sterile water, then placed on PDA at 25℃. Forty-eight isolates with the morphological characteristics of Colletotrichum ssp. were obtained by single-spore isolations (isolation frequency 42.5%). The fungal colonies on PDA were dense with white mycelia on the edges, and yellowish-white on the reverse side. The conidia were transparent, cylindrical, smooth-walled, and 6.8 to 17.5 × 3.8 to 6.5 µm. Two isolates (YNH-1 and YNH-2) were used for DNA extraction. The ribosomal internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin 2 (TUB2) regions were amplified by PCR. The PCR primers in this study were as described previously (Weir et al. 2012). The sequences of both isolates were 100% identical, and all sequences showed >98% identity with Colletotrichum siamense in the GenBank. The sequences were deposited in GenBank (ITS, PP237394; ACT, PP265410; CAL, PP265411; GAPDH, PP265412; TUB2, PP265413). A phylogenetic tree was constructed by MEGA_v. 11.0.13 with the Maximum Likelihood (ML) method. The isolate YNH-1 and YNH-2 clustered with C. siamense DAR 76934 (97% bootstrap support) in the same branch. Pathogenicity tests were performed on the susceptible maize variety B73. Twelve healthy maize seedlings were inoculated with a conidial suspension (1×106 conidia/ml) of isolate YNH-1. All the seedlings were kept in an incubator at 26℃, with a 90% humidity and a 12 h light/dark cycle. After 5 days, yellow spots appeared on the leaves of the plants. The symptoms on inoculated leaves were similar to those observed in the field after 10 days, whereas no symptoms appeared in the control. The pathogen C. siamensis was re-isolated from the infected leaves, which fulfilled the Koch's postulates. C. siamense can cause leaf diseases on a wide range of hosts. It has been reported causing anthracnose on tea (Camellia sinensis) (Wang et al. 2016) and wax apple (Syzygium samarangense) (Yao et al. 2023) in Yunnan Province, China. To our knowledge, this is the first report of C. siamense causing yellow leaf spots on maize in China as well as a new host record for C. siamense causing leaf disease. However, how C. siamense spreads among different host plants in the region is still unknown. This study provides important information for epidemiological study and comprehensive management of yellow leaf spot on maize.

First Report of Colletotrichum cordylinicola Causing Anthracnose on Cordyline fruticosa in China.

Cordyline fruticosa is a shrub plant, commonly used in landscape, and distributed in the tropical regions of southern China. In September 2022, anthracnose symptoms were found on this species in Nanning, Guangxi, China. The disease incidence was between 30% to 80% and disease severity was 10% to 30% in five surveyed planting areas. The symptoms initially appeared as small, round, brown spots on leaves. As the disease developed, the lesions turned gray-white with brown borders and yellow halos. Some spots coalesced into larger irregular shapes and even leading to leaf blight. Small segments of the diseased tissues (3×3 mm) were cut from the leaves, surface-sterilized by dipping in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA). These plates were incubated at 28°C in the dark for 5 days. Ten fungal isolates with similar morphology were consistently isolated from these diseased tissues. The colonies on PDA were initially white with sparse aerial mycelia and turned pale orange with abundant orange conidial masses on the center after 8 days of culture. The reverse color was pale orange. No sclerotia or setae were found in culture. Conidia were single-celled, hyaline, straight, cylindrical with round ends, and 12.2 to 17.8 µm long (mean 14.9 µm) and 3.9 to 7.3 µm wide (mean 4.8 µm, n=50). The morphological characteristics of these isolates were similar to the Colletotrichum cordylinicola (Sharma et al., 2014). Genomic DNA of two isolates Z3 and Z4 generated from monospore culture was extracted using a fungal DNA extraction kit (Solarbio, Beijing, China). Partial sequences of internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, and BT2A/BT2B (Lin et al., 2022), respectively. All the sequences (GenBank accession nos. OQ509909, OQ509910, OQ658690, OQ658691, and OK649310 to OK649314) showed 99% to 100% identity with those of C. cordylinicola in GenBank database. A phylogenetic tree based on concatenated sequences of ITS, ACT, CHS-1, TUB, and GAPDH using maximum likelihood analysis by MEGA X software revealed that Z3 and Z4 clade with reference strains of C. cordylinicola (OJX010226 and MK935473). Based on morphological observation and multi-gene sequence analysis, the isolates were identified as C. cordylinicola (Phoulivong et al., 2010). To assess their pathogenicity, conidial suspensions (106 conidia/ml) of C. cordylinicola were inoculated onto 10 healthy living leaves wounded by slight puncturing (10 µl/wounded spot). Control leaves were treated with sterile water. All inoculated and control plants were maintained under high relative humidity (~90%) and 28℃ in a climate chamber. After 8 days, all the inoculated leaves showed brown lesions resembling natural symptoms, whereas the control group remained symptom-free. The same fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. C. cordylinicola is a species of the C. gloeosporioides complex (Weir et al., 2012). It has been reported to cause anthracnose on C. fruticosa in USA and Thailand (Phoulivong et al., 2010; Sharma et al., 2014). To our knowledge, this is the first report of C. cordylinicola causing anthracnose on C. fruticosa in China. Knowing the causal agent is essential to control the serious disease effectively.