Insights into the cumulative effect of Colletotrichum gloeosporioides and Fusarium acutatum causing anthracnose-twister disease complex of onion.

Colletotrichum is an important plant pathogenic fungi that causes anthracnose/-twister disease in onion. This disease was prevalent in the monsoon season from August to November months and the symptoms were observed in most of the fields. This study aimed to investigate the pathogenicity and cumulative effect, if any of Colletotrichum gloeosporioides and Fusarium acutatum. The pot experiment was laid out to identify the cause responsible for inciting anthracnose-twister disease, whether the Colletotrichum or Fusarium or both, or the interaction of pathogens and GA3. The results of the pathogenicity test confirmed that C. gloeosporioides and F. acutatum are both pathogenic. C. gloeosporioides caused twisting symptoms independently, while F.acutatum independently caused only neck elongation. The independent application of GA3 did not produce any symptoms, however, increased the plant height. The combined treatment of C. gloeosporioides and F. acutatum caused twisting, which enhanced upon interaction with GA3 application giving synergistic effect. The acervuli were found in lesions infected with C. gloeosporioides after 8 days of inoculation on the neck and leaf blades. Symptoms were not observed in untreated control plants. Koch's postulates were confirmed by reisolating the same pathogens from the infected plants.

Biocontrol potential of endophytic bacterium Bacillus altitudinis GS-16 against tea anthracnose caused by Colletotrichum gloeosporioides.

Background: As one of the main pathogens causing tea anthracnose disease, Colletotrichum gloeosporioides has brought immeasurable impact on the sustainable development of agriculture. Given the adverse effects of chemical pesticides to the environment and human health, biological control has been a focus of the research on this pathogen. Bacillus altitudinis GS-16, which was isolated from healthy tea leaves, had exhibited strong antagonistic activity against tea anthracnose disease. Methods: The antifungal mechanism of the endophytic bacterium GS-16 against C. gloeosporioides 1-F was determined by dual-culture assays, pot experiments, cell membrane permeability, cellular contents, cell metabolism, and the activities of the key defense enzymes. Results: We investigated the possible mechanism of strain GS-16 inhibiting 1-F. In vitro, the dual-culture assays revealed that strain GS-16 had significant antagonistic activity (92.03%) against 1-F and broad-spectrum antifungal activity in all tested plant pathogens. In pot experiments, the disease index decreased to 6.12 after treatment with GS-16, indicating that strain GS-16 had a good biocontrol effect against tea anthracnose disease (89.06%). When the PE extract of GS-16 treated mycelial of 1-F, the mycelial appeared deformities, distortions, and swelling by SEM observations. Besides that, compared with the negative control, the contents of nucleic acids, protein, and total soluble sugar of GS-16 group were increased significantly, indicating that the PE extract of GS-16 could cause damage to integrity of 1-F. We also found that GS-16 obviously destroyed cellular metabolism and the normal synthesis of cellular contents. Additionally, treatment with GS-16 induced plant resistance by increasing the activities of the key defense enzymes PPO, SOD, CAT, PAL, and POD. Conclusions: We concluded that GS-16 could damage cell permeability and integrity, destroy the normal synthesis of cellular contents, and induce plant resistance, which contributed to its antagonistic activity. These findings indicated that strain GS-16 could be used as an efficient microorganism for tea anthracnose disease caused by C. gloeosporioides.

A tau class glutathione S-transferase in tea plant, CsGSTU45, facilitates tea plant susceptibility to Colletotrichum camelliae infection mediated by jasmonate signaling pathway.

Tea plant [Camellia sinensis (L.) O. Kuntze], as one of the most important commercial crops, frequently suffers from anthracnose caused by Colletotrichum camelliae. The plant-specific tau (U) class of glutathione S-transferases (GSTU) participates in ROS homeostasis. Here, we identified a plant-specific GST tau class gene from tea plant, CsGSTU45, which is induced by various stresses, including C. camelliae infection, by analyzing multiple transcriptomes. CsGSTU45 plays a negative role in disease resistance against C. camelliae by accumulating H2 O2 . JA negatively regulates the resistance of tea plants against C. camelliae, which depends on CsGSTU45. CsMYC2.2, which is the key regulator in the JA signaling pathway, directly binds to and activates the promoter of CsGSTU45. Furthermore, silencing CsMYC2.2 increased disease resistance associated with reduced transcript and protein levels of CsGSTU45, and decreased contents of H2 O2 . Therefore, CsMYC2.2 suppresses disease resistance against C. camelliae by binding to the promoter of the CsGSTU45 gene and activating CsGSTU45. CsJAZ1 interacts with CsMYC2.2. Silencing CsJAZ1 attenuates disease resistance, upregulates the expression of CsMYC2.2 elevates the level of the CsGSTU45 protein, and promotes the accumulation of H2 O2 . As a result, CsJAZ1 interacts with CsMYC2.2 and acts as its repressor to suppress the level of CsGSTU45 protein, eventually enhancing disease resistance in tea plants. Taken together, the results show that the JA signaling pathway mediated by CsJAZ1-CsMYC2.2 modulates tea plant susceptibility to C. camelliae by regulating CsGSTU45 to accumulate H2 O2 .

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

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

Antifeedant, Antifungal Cryptic Polyketides with Six Structural Frameworks from Tea Endophyte Daldinia eschscholtzii Propelled by the Antagonistic Coculture with Phytopathogen Colletotrichum pseudomajus and Different Culture Methods.

The antagonistic coculture with tea phytopathogen Colletotrichum pseudomajus induces antifungal cryptic metabolites from isogenesis endophyte Daldinia eschscholtzii against tea phytopathogens. Sixteen new polyketides with six structural frameworks including ten cryptic ones, named coldaldols A-C (1-3), collediol (5), and daldinrins A-L (10-20 and 23), were found from the coculture of C. pseudomajus and D. eschscholtzii by different culture methods. The unique framework of compounds 11 and 12 featured a benzopyran-C7 polyketone hybrid, and compounds 13-16 were characterized by the novel benzopyran dimer. The structures were determined mainly by spectroscopic methods, including extensive one-dimensional (1D), two-dimensional (2D) NMR, high resolution electrospray ionisation mass spectroscopy (HRESIMS), ECD calculation, and single-crystal X-ray diffraction. The configuration of acyclic compounds 5 and 18 were determined by application of the universal NMR database. Most compounds showed significant antifungal activities against the tea pathogens C. pseudomajus and Alternaria sp. with MICs of 1-8 µg/mL. Compound 12 had stronger antifungal activity than that of positive drug nystatin. The ether bond at C-4 of the benzopyran derivative increased the antifungal activity. Compounds 4-9 and 11-23 showed antifeedant activities against silkworms with feeding deterrence indices of 15-100% at the concentration of 50 µg/cm2.

The Role of Fatty Acids from Plant Surfaces in the Infectivity of Colletotrichum fioriniae.

Aqueous extracts derived from flowers stimulate germination, secondary conidiation, and appressorial formation of various latent fruit rotting fungi. Even raindrops passing over flowers accumulate sufficient activity to influence the infectivity of fruit rotting fungi. Using a spore germination bioassay, high levels of bioactivity were found in chloroform extracts from plant tissues, implicating the nonpolar components of the cuticle. The fatty acid (FA) and fatty acid methyl ester (FAME) composition (C9-C20) of blueberry and cranberry tissues as well as aqueous flower extracts were characterized using a gas chromatography-mass spectrometry (GC-MS) method. The FAs and FAMEs found in the plant extracts were then tested for bioactivity using a spore germination bioassay. The C16:0 and C18:2 FAs and FAMEs, as well as the C18:0 FAME and the C20:0 FA, all stimulated appressorial formation while the C10:0 FA stimulated secondary conidiation. The C10:0 and C16:0 FAs were the only two bioactive components also identified from the aqueous floral extracts of both blueberry and cranberry and are therefore considered as contributors to the bioactivity observed in these extracts. The aqueous extracts from surfaces other than flowers showed little or no activity, and it is speculated that the movement of FAs may be related to the level of polymerization and cutin polyester development in flowers versus other plant organs. This study highlights the importance of the bloom period for infection and that the apparent effects on host susceptibility may therefore depend on the availability of specific FAs or combinations thereof.

Genomic analysis of Colletotrichum camelliae responsible for tea brown blight disease.

BACKGROUND: Colletotrichum camelliae, one of the most important phytopathogenic fungi infecting tea plants (Camellia sinensis), causes brown blight disease resulting in significant economic losses in yield of some sensitive cultivated tea varieties. To better understand its phytopathogenic mechanism, the genetic information is worth being resolved. RESULTS: Here, a high-quality genomic sequence of C. camelliae (strain LT-3-1) was sequenced using PacBio RSII sequencing platform, one of the most advanced Three-generation sequencing platforms and assembled. The result showed that the fungal genomic sequence is 67.74 Mb in size (with the N50 contig 5.6 Mb in size) containing 14,849 putative genes, of which about 95.27% were annotated. The data revealed a large class of genomic clusters potentially related to fungal pathogenicity. Based on the Pathogen Host Interactions database, a total of 1698 genes (11.44% of the total ones) were annotated, containing 541 genes related to plant cell wall hydrolases which is remarkably higher than those of most species of Colletotrichum and others considered to be hemibiotrophic and necrotrophic fungi. It's likely that the increase in cell wall-degrading enzymes reflects a crucial adaptive characteristic for infecting tea plants. CONCLUSION: Considering that C. camelliae has a specific host range and unique morphological and biological traits that distinguish it from other species of the genus Colletotrichum, characterization of the fungal genome will improve our understanding of the fungus and its phytopathogenic mechanism as well.

Antitumor and antibacterial activity of metabolites of endophytic Colletotrichum siamense isolated from coffee (Coffea arabica L. cv IAPAR-59).

Endophytic fungi produce a range of known metabolites and several others, not yet explored, which present important biological activities from the pharmaceutical and industrial perspective. Several studies have reported the diversity of endophytes in Coffea arabica plants, although few have been described in organic cultures. In the current paper, we describe the chemical profile of specialized metabolites in the ethyl acetate phase in a strain of the endophytic fungus Colletotrichum siamense associated with coffee (Coffea arabica L.) (Rubiaceae) and its potential against tumor cells and bacteria of medical and food importance. Cytotoxicity assays in tumor cells MCF-7 and HepG2/C3A were performed by MTT and microdilution in broth to evaluate the antibacterial action of metabolic extract. The antiproliferative assay showed promising results after 24 h of treatment, with 50% injunction concentrations for the two cell types. UHPLC-MS/MS analyses with an electrospray ionization source were used to analyze the extracts and identify compounds of species Colletotrichum siamense, which is still little explored as a source of active metabolites. Many of these compounds observed in the endophytic need to be chemically synthesized in industry, at high costs, while production by the fungus becomes a chemically and economically more viable alternative. Pyrocatechol, gentisyl alcohol, and alpha-linolenic acid, associated with different mechanisms of action against tumor cells, were detected among the main compounds. The extract of the endophytic fungus Colletotrichum siamense presented several compounds with pharmacological potential and antibacterial activity, corroborating its potential in biotechnological applications.

Advances in understanding the mechanism of resistance to anthracnose and induced defence response in tea plants.

The tea plant (Camellia sinensis) is susceptible to anthracnose disease that causes considerable crop loss and affects the yield and quality of tea. Multiple Colletotrichum spp. are the causative agents of this disease, which spreads quickly in warm and humid climates. During plant-pathogen interactions, resistant cultivars defend themselves against the hemibiotrophic pathogen by activating defence signalling pathways, whereas the pathogen suppresses plant defences in susceptible varieties. Various fungicides have been used to control this disease on susceptible plants, but these fungicide residues are dangerous to human health and cause fungicide resistance in pathogens. The problem-solving approaches to date are the development of resistant cultivars and ecofriendly biocontrol strategies to achieve sustainable tea cultivation and production. Understanding the infection stages of Colletotrichum, tea plant resistance mechanisms, and induced plant defence against Colletotrichum is essential to support sustainable disease management practices in the field. This review therefore summarizes the current knowledge of the identified causative agent of tea plant anthracnose, the infection strategies and pathogenicity of C. gloeosporioides, anthracnose disease resistance mechanisms, and the caffeine-induced defence response against Colletotrichum infection. The information reported in this review will advance our understanding of host-pathogen interactions and eventually help us to develop new disease control strategies.

Multi-Function of a New Bioactive Secondary Metabolite Derived from Endophytic Fungus Colletotrichum acutatum of Angelica sinensis.

In the current study we assessed a new crystallized compound, 5-(1-hydroxybutyl)-4-methoxy-3-methyl-2H-pyran-2-one (C-HMMP), from the endophytic fungus Colletotrichum acutatum residing in the medicinal plant Angelica sinensis for its in vitro antimicrobial, antibiofilm, antioxidant, antimalarial, and anti-proliferative properties. The promising compound was identified as C-HMMP through antimicrobial-guided fraction. The structure of C-HMMP was unambiguously confirmed by 2D NMR and HIRS spectroscopic analysis. Antimicrobial property testing of C-HMMP showed it to be effective against a variety of pathogenic bacteria and fungi with MICs ranging from 3.9 to 31.25 µg/ml. The compound displayed excellent antibiofilm activity against C. albicans, S. aureus, and K. pneumonia. Furthermore, the antimalarial and radical scavenging activities of C-HMMP were clearly dosede-pendent, with IC50 values of 0.15 and 131.2 µg/ml. The anti-proliferative activity of C-HMMP against the HepG-2, HeLa, and MCF-7 cell lines in vitro was investigated by MTT assay, revealing notable anti-proliferative activity with IC50 values of 114.1, 90, and 133.6 µg/ml, respectively. Moreover, C-HMMP successfully targets topoisomerase I and demonstrated beneficial anti-mutagenicity in the Ames test against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF). Finally, the compound inhibited the activity of α-glucosidase and α-amylase with IC50 values of 144.7 and 118.6 µg/ml, respectively. To the best of our knowledge, the identified compound C-HMMP was obtained for the first time from C. acutatum of A. sinensis, and this study demonstrated that C-HMMP has relevant biological significance and could provide better therapeutic targets against disease.

Baseline Sensitivity and Resistance Mechanism of Colletotrichum Isolates on Tea-Oil Trees of China to Tebuconazole.

Colletotrichum fungi could cause anthracnose, a destructive disease in tea-oil trees. The sterol demethylation inhibitor (DMI) tebuconazole has been widely used in controlling plant diseases for many years. However, the baseline sensitivity of Colletotrichum isolates on tea-oil trees to tebuconazole has not been determined. In this study, the sensitivity to tebuconazole of 117 Colletotrichum isolates from tea-oil trees of seven provinces in southern China was tested. The mean effective concentration resulted in 50% mycelial growth inhibition (EC50), 0.7625 µg/ml. The EC50 values of 100 isolates (83%) were lower than 1 µg/ml, and those of 20 isolates (17%) were higher than 1 µg/ml, which implied that resistance has already occurred in Colletotrichum isolates on tea-oil trees. The EC50 values of the most resistant and sensitive isolates (named Ca-R and Cc-S1, respectively) were 1.8848 and 0.1561 µg/ml, respectively. The resistance mechanism was also investigated in this study. A gene replacement experiment indicated that the CYP51A/B gene of resistant isolates Ca-R and Cf-R1 cannot confer Cc-S1 full resistance to DMI fungicides, although three single point mutants, Cc-S1CYP51A-T306A and Cc-S1CYP51A-R478K, exhibited decreased sensitivity to DMI fungicides. This result suggested that resistance of Colletotrichum isolates was partly caused by mutations in CYP51A. Moreover, the expression level of CYP51A/B was almost identical among Ca-R, Cf-R1, Cc-S1, and Cc-S1CYP51A point mutants, which indicated that the resistance was irrelevant to the expression level of CYP51A, and other nontarget-based resistance mechanisms may exist. Our results could help to guide the application of DMI fungicides and be useful for investigating the mechanism of resistance.

Identification and Fungicide Sensitivity of Colletotrichum spp. from Apple Flowers and Fruitlets in Brazil.

Glomerella leaf spot (GLS) and bitter rot (BR), caused by Colletotrichum spp., are major diseases on apple in southern Brazil. Among integrated pest management tools for disease management in commercial orchards, fungicides remain an important component. This study aimed to identify Colletotrichum spp. from cultivar Eva in Paraná state orchards; evaluate their in vitro sensitivity to cyprodinil, tebuconazole, iprodione, and fluazinam; and determine the baseline in vitro sensitivity of these isolates to benzovindiflupyr and natamycin. Most isolates belonged to Colletotrichum melonis and C. nymphaeae of the C. acutatum species complex. The two species varied in sensitivity to fluazinam and tebuconazole, but no variability was found for any other fungicide. The lowest 50% effective concentration (EC50) values of Colletotrichum spp. were observed for cyprodinil (mean EC50 < 0.02) and benzovindiflupyr (mean EC50 < 0.05); EC50 values were intermediate for fluazinam (mean EC50 < 0.33) and tebuconazole (mean EC50 < 0.14), and they were highest for natamycin (mean EC50 < 5.56) and iprodione (mean EC50 > 12). Cyprodinil and fluazinam are registered for use in Brazil for apple disease management but not specifically for GLS and BR. Tebuconazole is one of the few products registered for Colletotrichum spp. control in apples. In conclusion, flowers and fruitlets can serve as sources of inoculum for GLS and BR disease; C. acutatum was the predominant species complex in these tissues; cyprodinil and fluazinam applications may suppress GLS and BR; and benzovindiflupyr and natamycin warrant further investigation for GLS and BR disease control of apple due to comparably high in vitro sensitivity.

Do Colletotrichum gloeosporioides and Rhizopus stolonifer induce alkaloidal and antifungal responses in Annona muricata seedlings?

The benzylisoquinoline alkaloids of Annona muricata have been isolated, but their physiological or ecological role is unknown. The objective was to explore whether these secondary metabolites are involved in defense against phytopathogenic fungi. To do this, the alkaloidal response of 6-leaf seedlings of A. muricata was analyzed, previously inoculated with Colletotrichum gloeosporioides and Rhizopus stolonifer. Before and after inoculation, alkaloidal extracts of roots, stems, and leaves were obtained, and the antifungal activity was evaluated in vitro. The alkaloids anonaine, reticuline, nornuciferine, assimilobine, and coreximine were identified. C. gloeosporioides caused variable increases in the production of anonaine, reticuline and nornuciferine (10-1200%), while R. stolonifer only stimulated the increase of nornuciferin and anonaine (10%) in the stems and leaves. The alkaloidal extracts of inoculated seedlings increased the antifungal activity, both against the pathogen elicitor and against the second target pathogen. These findings suggest that the alkaloids participate in the antifungal defense mechanism.

Analysis of the Pathogenicity and Phylogeny of Colletotrichum Species Associated with Brown Blight of Tea (Camellia sinensis) in Taiwan.

Brown blight, a destructive foliar disease of tea, has become a highly limiting factor for tea cultivation in Taiwan. To understand the population composition of the causal agents (Colletotrichum spp.), the fungal diversity in the main tea-growing regions all over Taiwan was surveyed from 2017 to 2019. A collection of 139 Colletotrichum isolates was obtained from 14 tea cultivars in 86 tea plantations. Phylogenic analysis using the ribosomal internal transcribed spacer, glutamine synthetase gene, Apn2-Mat1-2 intergenic spacer, ß-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase genes together with morphological characterization revealed three species associated with brown blight of tea; namely, Colletotrichum camelliae (95.6% of all isolates), C. fructicola (3.7%), and C. aenigma (0.7%). This is the first report of C. aenigma in Taiwan. The optimal growth temperatures were 25°C for C. camelliae and 25 and 30°C for C. fructicola and C. aenigma. Although C. fructicola and C. aenigma were more adapted to high temperature, C. camelliae was the most pathogenic across different temperatures. Regardless of whether spore suspensions or mycelial discs were used, significantly larger lesions and higher disease incidences were observed for wounded than for nonwounded inoculation and for the third and fourth leaves than for the fifth leaves. Wounded inoculation of detached third and fourth tea leaves with mycelial discs was found to be a reliable and efficient method for assessing the pathogenicity of Colletotrichum spp. within 4 days. Preventive application of fungicides or biocontrol agents immediately after tea pruning and at a young leaf stage would help control the disease.

Phosphate solubilization and indole-3-acetic acid (IAA) produced by Colletotrichum gloeosporioides and Aspergillus fumigatus strains isolated from the rhizosphere of Dillenia indica L.

Plants form associations with different microbes; some promote their growth and protect from biotic and abiotic stresses in different ways. However, the biological role of fungi associated with the rhizosphere of medicinal plants is not well explored. In the present study, Colletotrichum gloeosporioides, and Aspergillus fumigatus isolated from the rhizosphere of Dillenia indica were screened for their phosphate solubilization and indole-3-acetic acid (IAA) production potential. The selected fungal strains were identified by macroscopic, microscopic, and molecular characteristics. Phosphate solubilization was qualitatively and quantitatively evaluated using Pikovskaya's (PVK) agar and PVK broth medium using different substrates such as AlPO4, Ca3(PO4)2, and FePO4. Colletotrichum gloeosporioides and Aspergillus fumigatus with respect to the phosphate source showed solubilization index (SI) of 1.7 ± 0.03 and 2.1 ± 0.04, and solubilized phosphate up to 138.8 ± 0.058 µg/mL and 121.6 ± 0.062 µg/mL. These fungal strains are also good producers of IAA and significantly enhance the growth of Vigna radiata and Cicer arietinum seedlings. This is the first report on A. fumigatus and C. gloeosporioides from the rhizosphere of Dillenia indica and their phosphate solubilization and IAA production ability.

Genome sequencing of Colletotrichum gloeosporioides ES026 reveals plausible pathway of HupA.

BACKGROUND: Colletotrichum gloeosporioides ES026, isolated as an endophytic fungal strain, was found to produce the important medicinal compound HuperzineA (HupA). In a genetic context, ES026 showed potential in elucidating the biosynthetic pathway of HupA. METHODS AND RESULTS: The ES026 strain was sequenced using de-novo Illumina sequencing methods in this study. Assembling the cleaned data resulted in 58,594,804bp, consisting of 404 scaffolds. The G + C mol % content of this genome was 52.53%. The genome progressive-alignment with other 4 Colletotrichum strains revealed that ES026 showed closer relation with 030206, SMCG1#C and Nara gc5. More than 60 putative biosynthetic clusters were predicted with the fungal version antiSMASH4.0 program. More than 33 types I polyketide-related biosynthetic gene clusters were distributed, containing PKS and PKS-NRPS (polyketide-nonribosomal peptides) hybrid gene clusters. Another 8 NRPS biosynthetic gene clusters were distributed among the genome of ES026. The prenyltransferases, probably involved in aromatic prenyl-compounds and terpenoid biosynthesis, were analyzed using bioinformatics tools like MEGA. CONCLUSION: We predicted a new possible biosynthetic pathway for the HupA from the pipecolic acid, based on the published HupA biosynthesis proposed pathway, the biosynthesis and pipecolic acid-derived compounds. We hypothesize that a hybrid PKS-NRPS mega-enzyme was probably involved in the biosynthesis of HupA with the pipecolic acid, the building block of rapamycin, as a HupA precursor. The rapamycin is produced from a polyketide biosynthesis pathway, and the domain incorporating the pipecolic acid is studied.

Retromer subunit, CfVps35 is required for growth development and pathogenicity of Colletotrichum fructicola.

BACKGROUND: Tea oil is widely used as edible oil in China, which extracted from the seeds of Camellia oleifera. In China, the national oil-tea camellia planting area reached 4.533 million hectares, the output of oil-tea camellia seed oil was 627 000 tons, and the total output value reached 18.3 billion dollars. Anthracnose is the common disease of Ca. oleifera, which affected the production and brought huge economic losses. Colletotrichum fructicola is the dominant pathogen causing anthracnose in Ca. oleifera. The retromer complex participates in the intracellular retrograde transport of cargos from the endosome to the trans-Golgi network in eukaryotes. Vacuolar protein sorting 35 is a core part of the retromer complex. This study aimed to investigate the role of CfVps35 in C. fructicola. RESULTS: The CfVPS35 gene was deleted, resulting in reduced mycelial growth, conidiation, and response to cell wall stresses. Further analysis revealed that CfVps35 was required for C. fructicola virulence on tea oil leaves. In addition, the ΔCfvps35 mutant was defective in glycogen metabolism and turgor during appressorium development. CONCLUSION: This study illustrated that the crucial functions of CfVps35 in growth, development, and pathogenicity.

Identification and Observation of Infection Processes of Colletotrichum Species Associated with Pearl Plum Anthracnose in Guangxi, China.

Pearl plum (Prunus salicina Lindl.) is mainly cultivated in Tian'e County in Guangxi Province, southern China. Anthracnose is a devastating disease on pearl plum, causing extensive leaf blight. Diseased leaves were sampled from 21 orchards in Tian'e County. Isolates were first screened for ones resembling Colletotrichum, and 21 representative isolates were selected for sequencing of portions of the ribosomal internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 genes (ApMAT), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), and ß-tubulin 2 (TUB2). Based on colony, conidial, and appressorial morphology and sequence analyses, the Colletotrichum isolates associated with pearl plum anthracnose were identified as four species: Colletotrichum fructicola (16 isolates), C. gloeosporioides (3 isolates), C. cigarro (1 isolate), and C. siamense (1 isolate). The results of pathogenicity tests showed that isolates of all four species were pathogenic to wounded leaves of pearl plum seedlings. In this study, we microscopically observed the infection processes of isolates of these four species on attached pearl plum leaves. For C. cigarro and C. siamense, the entire infection processes took 120 h; for C. fructicola and C. gloeosporioides, it only took 72 h. This is the first report of C. fructicola and C. cigarro causing anthracnose on pearl plum worldwide, and also the first report of C. siamense causing anthracnose on pearl plum in China.

The CfMcm1 Regulates Pathogenicity, Conidium Germination, and Sexual Development in Colletotrichum fructicola.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola, is a severe disease worldwide on apple, causing defoliation, leaf and fruit spot, and substantial yield loss. However, little is known about its molecular mechanisms of pathogenesis. Previous transcriptome analysis revealed that a transcription factor, CfMcm1, was induced during leaf infection. In the present work, expression pattern analysis verified that the CfMcm1 gene was strongly expressed in conidia and early infection. Phenotypic analysis revealed that the gene deletion mutant ΔCfMcm1 lost pathogenicity to apple leaves by inhibiting conidial germination and appressorium formation. In addition to appressorium-mediated pathogenicity, ΔCfMcm1 colonization and hyphal extension in wounded apple fruit was also reduced, and conidial germination mode and conidial color were altered. ΔCfMcm1 displayed impairment of cell wall integrity and response to stress caused by oxidation, osmosis, and an acid environment. Furthermore, the deletion mutant produced fewer and smaller perithecia and no ascospores. In contrast, melanin deposition in mycelia of ΔCfMcm1 was strengthened. Further comparative transcriptome and quantitative PCR analysis revealed that CfMcm1 modulated expression of genes related to conidial development (CfERG5A, CfERG5B, CfHik5, and CfAbaA), appressorium formation (CfCBP1 and CfCHS7), pectin degradation (CfPelA and CfPelB), sexual development (CfMYB, CfFork, CfHMG, and CfMAT1-2-1), and melanin biosynthesis (CfCmr1, CfPKS1, CfT4HR1, CfTHR1, and CfSCD1). Our results demonstrated that CfMcm1 is a pivotal regulator possessing multiple functions in pathogenicity, asexual and sexual reproduction, and melanin biosynthesis.

Characterization of the WRKY gene family in Akebia trifoliata and their response to Colletotrichum acutatum.

BACKGROUND: Akebia trifoliata, belonging to the Lardizabalaceae family, is a well-known Chinese traditional medicinal plant, susceptible to many diseases, such as anthracnose and powdery mildew. WRKY is one of the largest plant-specific transcription factor families and plays important roles in plant growth, development and stress response, especially in disease resistance. However, little was known about the numbers, characters, evolutionary relationship and expression of WRKY genes in A. trifoliata in response to plant disease due to lacking of A. trifoliata genome. RESULTS: A total of 42 putative AktWRKY genes were identified based on the full-length transcriptome-sequencing data of A. trifoliata. Then 42 AktWRKY genes were divided into three major groups (Group I-III) based on the WRKY domains. Motif analysis showed members within same group shared a similar motif composition, implying a functional conservation. Tissue-specific expression analysis showed that AktWRKY genes could be detected in all tissues, while few AktWRKY genes were tissue specific. We further evaluated the expression of AktWRKY genes in three varieties in response to Colletotrichum acutatum by qRT-PCR. The expression patterns of AktWRKY genes were similar between C01 and susceptible variety I02, but distinctly different in resistant variety H05. In addition, it showed that more than 64 percentages of AktWRKY genes were differentially expressed during fungal infection in I02 and H05. Furthermore, Gene ontology (GO) analysis showed that AktWRKY genes were categorized into 26 functional groups under cellular components, molecular functions and biological processes, and a predicted protein interaction network was also constructed. CONCLUSIONS: Results of bioinformation analysis and expression patterns implied that AktWRKYs might play multiple function in response to biotic stresses. Our study could facilitate to further investigate the function and regulatory mechanism of the WRKY in A. trifoliata during pathogen response.