Hyphal Morphology Research Articles

Analysis of the effect of Bacillus velezensis culture filtrate on the growth and proteome of Cladobotryum mycophilum

Cladobotryum mycophilum, the causative agent of cobweb disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Cobweb disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant Cladobotryum strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of C. mycophilum to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. Exposure of 48 hr C. mycophilum cultures to 25% v/v 96 hr B. velezensis culture filtrate resulted in a 57% reduction in biomass (P < 0.0002), a disruption in hyphal structure and morphology, and the appearance of aurofusarin, a secondary metabolite which is a known indicator of oxidative stress, in culture medium. Proteomic analysis of B. velezensis culture filtrate revealed the presence of peptidase 8 (subtilisin), peptide deformylase and probable cytosol aminopeptidase which are known to induce catalytic activity. Characterisation of the proteomic response of C. mycophilum following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (ISWI chromatin-remodelling complex ATPase ISW2 (+24 fold), carboxypeptidase Y precursor (+3 fold) and calmodulin (+2 fold). There was also a decrease in the abundance of proteins associated with transcription (40 S ribosomal protein S30 (−26 fold), 40 S ribosomal protein S21 (−3 fold) and carbohydrate metabolism (l-xylulose reductase (−10 fold). The results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of C. mycophilum and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms.

SUN-Family Protein UvSUN1 Regulates the Development and Virulence of Ustilaginoidea virens

Ustilaginoidea virens, the causal agent of rice false smut disease, is an important plant pathogen that causes severe quantitative and qualitative losses in rice worldwide. UvSUN1 is the only member of Group-I SUN family proteins in U. virens. In this work, the role of UvSUN1 in different aspects of the U. virens biology was studied by phenotypic analysis of Uvsun1 knockout strains. We identified that UvSUN1 was expressed during both conidial germination and the infection of rice. Disruption of the Uvsun1 gene affected the hyphal growth, conidiation, morphology of hyphae and conidia, adhesion and virulence. We also found that UvSUN1 is involved in the production of toxic compounds, which are able to inhibit elongation of the germinated seeds. Moreover, RNA-seq data showed that knockout of Uvsun1 resulted in misregulation of a subset of genes involved in signal recognition and transduction system, glycometabolism, cell wall integrity, and secondary metabolism. Collectively, this study reveals that Uvsun1 is required for growth, cell wall integrity and pathogenicity of U. virens, thereby providing new insights into the function of SUN family proteins in the growth and pathogenesis of this pathogen.

Large Scale Cultivation of Bacillus velezensis CE 100 and Effect of Its Culture on Control of Citrus Melanose Caused by Diaporthe citri

Diaporthe citri is an important fungal pathogen which causes melanose in various citrus growing regions of the world. In our study, control of D. citri was performed by using effective biocontrol agent, Bacillus velezensis CE 100. Mycelial growth of D. citri was highly restricted by strain CE 100 in dual culture plates. Our study demonstrates large-scale inoculation of strain CE 100 in non-sterilized PB medium. In addition, bacterial culture filtrate (BCF) of strain CE 100 from the large-scale culture showed better growth inhibition of D. citri than those from small-scale culture in the laboratory. Moreover, microscopic observation revealed that hyphal morphology of the pathogen was significantly affected by BCF, probably because of various cell-wall degrading enzymes and metabolites produced by strain CE 100. In addition, 50% BCF of strain CE 100 inhibited D. citri conidial germination by over 80%. Treatments, applied by foliar spray with fungicide and strain CE 100 culture, significantly lowered melanose disease incidence more than did control. The results of this study indicate that large-scale culture of strain CE 100 was effective for control of D. citri by means of adequate active metabolites and cells. Therefore, we demonstrate biocontrol applicability of B. velezensis CE 100 on control of citrus melanose by developing a low-cost PB medium using affordable large-scale fermenter. Effect of different treatments on melanose disease incidence on leaves of mandarin plants at 20 days after inoculation with D. citri. Statistical comparison between treatments was done by least significant difference (LSD) test at p < 0.05 level. Means with the same letter are not significantly different from each other. *Con- Control, CE 100- Treatment with B. velezensis CE 100 culture, F- Treatment with fungicide

Active compound identification by screening 33 essential oil monomers against Botryosphaeria dothidea from postharvest kiwifruit and its potential action mode

The antifungal activity of postharvest kiwifruit against the pathogen Botryosphaeria dothidea was evaluated for 33 essential oil monomers. The possible mechanism for the known active compounds were further assessed in this study. The results show all the EO components exhibit inhibitory effects on the pathogen to different degrees except for Farnesol. Carbon chain length and C2-C3 double bonds had a great effect on the antifungal activities of aldehydes. Of all of these, carvacrol had the strongest antifungal activity with EC50 of 12.58 μL/L and EC90 of 22.08 μL/L. Carvacrol also exhibits significant inhibitory effects on the pathogen, both in vivo and in vitro. Carvacrol evidently alters the hyphal morphology of B. dothidea and severely damages cell membrane and inhibits the formation of lipid components on the membrane. As cell membrane permeability increases, intracellular homeostasis including ion and biomacromolecules were destroyed by carvacrol. Furthermore, carvacrol appears to significantly inhibit mitochondrial activity and respiration rates, resulting in cell death of B. dothidea. Our results provide evidence that carvacrol could be a very useful compound for controlling postharvest rot soft in kiwifruit.

The Fvclp1 gene regulates mycelial growth and fruiting body development in edible mushroom Flammulina velutipes.

Fruiting bodydevelopment inAgaricomycetesrepresents the most complex and unclear process in the fungi.Mating type pathways (A and B)and transcription factors are important regulators in the sexual development of mushrooms. It is known that clampless1 (clp1) is an additional gene that participate under the homeodomain (HD) genes in the matA pathway and clp1 inactivation blocks clamps formation in Coprinopsis cinerea. In this study we identified and analyzed a homologous Fvclp1 gene in the edible mushroom Flammulina velutipes. The coding sequence of the Fvclp1 was 1011bp without intron interruption, encoding a protein of 336 amino acids. To exhibit the role of Fvclp1 in clamp development and fruiting body formation, knockdown and overexpression mutants were prepared. No significant difference was observed in the monokaryotic hyphal morphology of overexpression and knockdown transformants. In the dikaryotic hyphae from the compatible crossings between the wild-type L22 strain and Fvclp1 knockdown or overexpression mutants, clamp connections developed. However, knockdown mutants could generate fewer fruiting bodies than the wild-type strain. On the contrary, reduced mycelial growth rate but improved fruiting ability was observed in the dikaryotic Fvclp1 overexpression mutants as compared to the wild-type strain. These results indicate that Fvclp1 is necessary and actively involved in fruiting body development in F. velutipes. Overall, these findings suggest that further studies on the function of Fvclp1 would advance our understanding of sexual reproduction and fruiting body development in edible mushrooms.

Synthesis, Bioactivity, and QSAR Study of 3,4-Dichlorophenyl Isoxazole-Substituted Stilbene Derivatives against the Phytopathogenic Fungus Botrytis cinerea.

Gray mold, caused by Botrytis cinerea, is one of the most destructive fungal diseases in crops, responsible for significant economic losses. In search of natural product-based fungicides, we designed and synthesized a series of novel 3,4-dichlorophenyl isoxazole-substituted stilbene derivatives, and their in vivo antifungal activities against B. cinerea were evaluated. The results indicated that some of the target molecules demonstrated remarkable efficiency for the control of tomato gray mold. In particular, compound 5r displayed the highest fungicidal potency with an inhibition rate of 56.11% comparable to that of positive control boscalid (66.96%). Moreover, a hologram quantitative structure-activity relationship (HQSAR) model with good predictive capability was developed to provide in-depth insight into the activity profiles of these compounds. Preliminary mechanism studies suggested that compound 5r might exert its antifungal effect by changing hyphal morphology and increasing the membrane permeability. The present study contributes to the development of natural stilbene derivatives as alternative bioactive agents against B. cinerea.

Potential roles of essential oils from the flower, fruit and leaf of Citrus medica L. var. sarcodactylis in preventing spoilage of Chinese steamed bread

This study investigated the composition and antifungal activities of the essential oil (EO) of golden finger citron (Citrus medica L. var. sarcodactylis, GFC). The EOs obtained by hydrodistillation were characterized by GC-MS and GC-FID. The study revealed that the inhibition zone diameter (IZD) of EOs against mould growth on Chinese steamed bread (CSB) was 11.33–90.00 mm, the minimum inhibition concentration (MIC) was 0.37–6 μL/mL and that EOs extended the shelf life of CSB by 3–13 days in the vacuum package. Scanning electron microscopy (SEM) revealed the EOs altered the hyphal morphology of the moulds at the MIC. Sixteen bioactive compounds were identified based on their antifungal activities. The results demonstrate that the EOs of GFC have the potential to act as plant-based biopreservatives in CSB.

Isolation and Characterization of Nickel-Tolerant Trichoderma Strains from Marine and Terrestrial Environments.

Nickel contamination is a serious environmental issue that requires immediate action. In this study, 23 strains of Trichoderma were isolated from terrestrial and marine environments and identified using a polyphasic approach of morphological characterization and ITS gene sequence analysis. The Trichoderma strains were tested for their tolerance and biosorption of nickel. Our results showed the growth of all Trichoderma strains on Trichoderma Selective Medium (TSM) with 50–1200-ppm nickel, indicating their tolerance of this heavy metal even at a relatively high concentration. Six Trichoderma strains (three isolated from terrestrial substrates and three from marine substates) had the highest radial growth on TSM with 50-ppm Ni. Among these fungal isolates, Trichoderma asperellum (S03) isolated from soil exhibited the best growth after 2 days of incubation. For the biosorption of nickel, the accumulation or uptake efficiency by the six selected Trichoderma was determined in Potato Dextrose Broth (PDB) supplemented with 50-ppm Ni using a Flame Atomic Absorption Spectrophotometer (AAS). The percent uptake efficiency of the three strains of T. asperellum (S03, S08, and LL14) was computed to be up to 66%, while Trichoderma virens (SG18 and SF22) and Trichoderma inhamatum (MW25) achieved up to 68% uptake efficiency. Observation of the Trichoderma strains with Scanning Electron Microscopy (SEM) before and after the absorption of nickel showed very minimal damage on the hyphal and conidial surface morphology, but changes in the colonial characteristics were observed. Our study highlighted the potential of terrestrial and marine strains of Trichoderma for the bioremediation of nickel pollution.

Insight into the substrate-dependent anti-aflatoxigenic effects of nanosized ZnO film: Electron transfer directed oxidative stress mechanisms

Exploring new anti-aflatoxigenic materials and their mechanisms are critical to reduce the prevalence of drug-resistant fungi and the contamination of aflatoxins. Zinc oxide nanoparticles (ZnONPs) are promising antifungal candidates but supporting substrates generally affect their antifungal activities. In this study, ZnONPs with a three-dimensional flower-like hierarchical microstructure bound to different substrates as anti-aflatoxigenic composites were prepared using a facile deposition method. It was found that ZnO nanocomposites showed the substrate-dependent anti-aflatoxigenic activities. The antifungal activities of ZnO films toward A. flavus growth and aflatoxin B1 production decreased significantly in the order ZnO@Zn＞ZnO@Sn＞ZnO@Steel＞ZnO@Glass. The electrical conductivity of the substrate should play an important role for antifungal response. When compared with ZnO@Sn and ZnO@Steel, the conductivity value of ZnO@Zn was 2.07-fold and 14.84-fold of them, respectively. The higher the electrical conductivity of the substrate, the better the anti-aflatoxigenic efficiency of the composite. Such anti-aflatoxigenic activity was also due to ROS generation through electron transfer between fungi and the ZnO-substrate system, which could provoke the strength of intracellular oxidative stress. This mechanism was further confirmed using several assays such as hyphal morphology analysis, Zn2+ release, ROS evaluation, lipid peroxidation and antioxidant response. Collectively, improvement in knowledge regarding anti-aflatoxigenic performance of ZnONPs can help develop novel and effective strategies to reduce fungi growth and aflatoxin contamination in the food field.

Candidalysin delivery to the invasion pocket is critical for host epithelial damage induced by Candida albicans.

The human pathogenic fungus Candida albicans is a frequent cause of mucosal infections. Although the ability to transition from the yeast to the hypha morphology is essential for virulence, hypha formation and host cell invasion per se are not sufficient for the induction of epithelial damage. Rather, the hypha-associated peptide toxin, candidalysin, a product of the Ece1 polyprotein, is the critical damaging factor. While synthetic, exogenously added candidalysin is sufficient to damage epithelial cells, the level of damage does not reach the same level as invading C. albicans hyphae. Therefore, we hypothesized that a combination of fungal attributes is required to deliver candidalysin to the invasion pocket to enable the full damaging potential of C. albicans during infection. Utilising a panel of C. albicans mutants with known virulence defects, we demonstrate that the full damage potential of C. albicans requires the coordinated delivery of candidalysin to the invasion pocket. This process requires appropriate epithelial adhesion, hyphal extension and invasion, high levels of ECE1 transcription, proper Ece1 processing and secretion of candidalysin. To confirm candidalysin delivery, we generated camelid VH Hs (nanobodies) specific for candidalysin and demonstrate localization and accumulation of the toxin only in C. albicans-induced invasion pockets. In summary, a defined combination of virulence attributes and cellular processes is critical for delivering candidalysin to the invasion pocket to enable the full damage potential of C. albicans during mucosal infection. TAKE AWAYS: Candidalysin is a peptide toxin secreted by C. albicans causing epithelial damage. Candidalysin delivery to host cell membranes requires specific fungal attributes. Candidalysin accumulates in invasion pockets created by invasive hyphae. Camelid nanobodies enabled visualisation of candidalysin in the invasion pocket.

Competency of Clove and Cinnamon Essential Oil Fumigation against Toxigenic and Atoxigenic Aspergillus flavus Isolates

Aspergillus flavus is a frequent contaminant of maize grain. We isolated this fungus, determined the colony morphology and species (by internal transcribed spacer sequencing) and measured the aflatoxin content. The selected A. flavus fungi were placed into two groups, toxigenic and atoxigenic; both appeared similar morphologically, except that the atoxigenic group lacked sclerotia. An essential oil fumigation test with clove and cinnamon oils as antifungal products was performed on fungal conidial discs and fungal colonies in Petri plates. Cinnamon oil at 2.5 to 5.0 μL/plate markedly inhibited the mycelial growth from conidial discs of both strains, whereas clove oil showed less activity. The oils had different effects on fungal mycelia. The higher clove fumigation doses of 10.0 to 20.0 μL/plate controlled fungal growth, while cinnamon oil caused less inhibition. Compared with atoxigenic groups, toxigenic A. flavus responded stably. Within abnormal A. flavus hyphae, the essential oils degenerated the hyphal morphology, resulting in exfoliated flakes and shrinkage, which were related to fungal membrane injury and collapse of vacuoles and phialide. The treatments, especially those with cinnamon oil, increased the electroconductivity, which suggested a weak mycelium membrane structure. Moreover, the treatments with essential oils reduced the ergosterol content in mycelia and the aflatoxin accumulation in the culture broth. The fumigations with clove and cinnamon oils inhibited the development of both conidia and colonies of A. flavus in dose-dependent manners.

The Success of Topical Treatment of Onychomycosis Seems to Be Influenced by Fungal Features.

Aim To evaluate the topical treatment of onychomycosis using a 10% hydroalcoholic propolis extract (PE) in two aleatorily chosen patients and analyze possible risk factors from hosts including some particularities of the isolated fungi that may justify the outcomes achieved. Materials and Methods A topical treatment, with PE, was started in two cases of toe onychomycosis due to T. rubrum. The in vitro PE antifungal activity against these isolates was confirmed. Moreover, the ability of the fungi to infect the human nail was evaluated also in an ex vivo study, analyzed by histopathology. Results Within four months, both patients showed evident improvement, but with different outcomes. The possible host-related risk factors justifying the poorer outcome in patient 1 include a longer duration time of onychomycosis (50 years). Some particularities in the T. rubrum strain isolated from this patient in relation to that found in patient 2 were observed: (1) the hypha morphology suggesting a major adaptation of the fungus to the host; (2) a 16 times greater propolis concentration was required in vitro; and (3) a faster ability to start a growth using the nail as the only nutritional source. Additionally, this isolate was more efficient in producing a biofilm on the nail surface. Conclusions A partial clinical and complete mycological cure for the two patients was achieved after four months of PE daily use. Despite a complete recovery, a different outcome was observed between both cases. A more persistent onychomycosis, added to greater fungal potential to produce biofilm on the nail, seems to influence greatly the success of a topical treatment with PE.

ChSch9 is required for infection-related morphogenesis and pathogenicity in Colletotrichum higginsianum

Colletotrichum higginsianum is an economically important anthracnose pathogen of crucifers that causes losses worldwide. For comprehensive control of anthracnose disease, it is essential to understand the mechanisms of pathogenicity in C. higginsianum. The Ser/Thr kinase Sch9 belonging to the AGC (protein kinase A, G, and C) kinase family, commonly found in eukaryotes, plays a vital role in various cell signalling pathways and development. However, it has not been studied in Colletotrichum species. Three domains, Protein kinase C conserved region 2 (C2), Serine/Threonine protein kinase catalytic (S_TKc) and Extension to Ser/Thr-type protein kinases (S_TK_X), were identified in the C. higginsianum Sch9 (ChSch9) protein. Phylogenetic analysis indicated that ChSch9 is closely related to the AGC kinase family, which is conserved in fungi. Deletion of ChSch9 caused defects in hyphal morphology, growth and conidiation. In addition, deletion of ChSch9 was associated with reduced tolerance to hyperosmotic, cell wall and oxidative stresses in C. higginsianum, and ChSch9 deletion mutants were more tolerant to heat. Furthermore, we found that ChSch9 might be involved in the pyruvate–acetaldehyde fermentation pathway. Importantly, deletion of ChSch9 led to a reduction in appressoria and infective hyphae formation, resulting in defects in virulence. Collectively, the results of this study indicate that ChSch9 is required for infection-related morphogenesis and virulence in C. higginsianum.

Cellular uptake of chitosan and its role in antifungal action against Penicillium expansum

Chitosan has wide-spectrum antimicrobial activity but knowledge of its antifungal mechanism is still incomplete. In this study, transcriptome of Penicillium expansum upon chitosan treatment was analyzed by RNA-Seq. KEGG enrichment analysis revealed that endocytosis as well as other physiological pathways was regulated by chitosan treatment. Clathrin adaptor protein mu-subunit (PeCAM) gene, which encodes a protein associated with clathrin-dependent endocytosis, was up-regulated after chitosan treatment. Deletion of PeCAM resulted in changes of conidial, hyphal and colonial morphology. Confocal microscopy images of the distribution of fluorescein isothiocyanate-labeled chitosan confirmed cellular internalization of chitosan. However, deletion of PeCAM almost completely blocked uptake of chitosan into fungal cells and ΔPeCAM mutant exhibited less sensitivity to chitosan compared with wild type, suggesting that chitosan uptake is mediated by clathrin-dependent endocytosis and internalized chitosan also plays an important role in its antifungal activity. Collectively, our results provide a new insight into the antifungal mechanism of chitosan.

Fungal interactions induce changes in hyphal morphology and enzyme production

ABSTRACT In nature, species interacts/competes with one other within their surrounding for food and space and the type of interactions are unique to each species. The interacting partners secrete different metabolites, which may have high importance in human welfare. Fungal–fungal interactions are complex mechanisms that need better understanding. Here, 14 fungal isolates were facilitated in 105 possible combinations to interact on potato dextrose agar. Morphologically, no changes were observed when the same fungal isolates were allowed to interact within them. However, 10 interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Contrastingly, 35 interactions resulted into complete replacement as one of the fungi was inhibited by rapid growth of the other fungus. In 46 interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. To study in details about the barrage formation, two fungal interactions were taken (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes in the hyphal growth during interaction were observed. There was significant increase in the enzymatic activities including cellulase, xylanase and chitinase during in-vitro fungal–fungal interaction, suggesting the importance of such interactions for commercial enzyme production.

Antibacterial Performance of a Streptomyces spectabilis Strain Producing Metacycloprodigiosin.

After separation of bacterial colonies on solid plates, purification, and screening through the agar cup-plate method, an antibiotic-resistant bacterial isolate was obtained, and named strain L20190601, the 16S rRNA gene sequence data of strain L20190601 to GenBank, NCBI have provided GenBank accession number MW931615. 16S rRNA gene sequencing revealed that this isolate was highly similar to a number of Streptomyces species. Among them, the homology with S. spectabilis was the highest, reaching 99.9, together with curved hyphal morphology and biochemical tests, allowed us to identify strain L20190601 as S. spectabilis. The red pigment produced by S. spectabilis strain L20190601 was structurally identified. An acid-base color reaction assay showed that when this pigment was dissolved in a solution at pH 3.0 and 9.0, the color of the solution was red and yellow, respectively. In addition, the analysis of absorption spectra revealed that at pH 8.0 and 3.0, the maximum absorption peaks were at 466 and 531nm, respectively. These results are consistent with the spectral absorption characteristics of metacycloprodigiosin reported in the literature. Moreover, the retention time of purified pigments was identical to those of standard metacycloprodigiosin solutions. Mass spectrometry analysis revealed that the molecular weight of the red compound was 392.2 [M + H]+. Finally, metacycloprodigiosin was found to be effective against eight clinically common pathogens: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus typhi, Candida albicans, and Trichophyton rubrum. In summary, metacycloprodigiosin exhibited strong antibacterial activity and a broad antibacterial spectrum, and thus is a promising compound for the development of a new type of antibacterial drug.

Coupled Shifts in Ectomycorrhizal Communities and Plant Uptake of Organic Nitrogen Along a Soil Gradient: An Isotopic Perspective

Plants associating with mutualistic ectomycorrhizal (ECM) fungi may directly obtain nitrogen (N) bound in soil organic matter (N-SOM). However, the contribution of N-SOM to plant growth under field conditions remains poorly constrained. We tested the hypothesis that turnover in ECM communities along soil inorganic N gradients mediates a functional transition from plant reliance on N-SOM in low inorganic N soils, to primarily inorganic N uptake in inorganic N-rich condition soils. We quantified the δ 15 N of Q. rubra foliage and roots, organic and inorganic soil N pools, and used molecular sequencing to characterize ECM communities, morpho-traits associated with N-foraging, and a community aggregated sporocarp δ 15 N. In support of our hypothesis, we document the progressive enrichment of root and foliar δ 15 N with increasing soil inorganic N supply; green leaves ranged from − 5.95 to 0.16‰ as the supply of inorganic N increased. ECM communities inhabiting low inorganic N soils were dominated by the genus Cortinarius, and other fungi forming hyphal morphologies putatively involved in N-SOM acquisition; sporocarp estimates from these communities were enriched (+ 4‰), further supporting fungal N-SOM acquisition. In contrast, trees occurring in high inorganic N soils hosted distinct communities with morpho-traits associated with inorganic N acquisition and depleted sporocarps (+ 0.5‰). Together, our results are consistent with apparent tradeoffs in the foraging cost and contribution of N-SOM to plant growth and demonstrate linkages between ECM community composition, fungal N-foraging potential and foliar δ 15 N. The functional characteristics of ECM communities represent a mechanistic basis for flexibility in plant nutrient foraging strategies. We conclude that the contribution of N-SOM to plant growth is likely contingent on ECM community composition and local soil nutrient availability.

Inhibitory effects and mechanisms of vanillin on gray mold and black rot of cherry tomatoes

Vanillin is a natural antimicrobial agent; however, there are few reports on its antifungal effect on postharvest pathogenic fungi. This study aimed to investigate the in vivo and in vitro antifungal activities of vanillin against gray mold (caused by B. cinerea) and black rot (caused by A. alternata) of cherry tomato fruit and to explain its possible mechanism of action. Vanillin strongly inhibits Botrytis cinerea and Alternaria alternata mycelial growth, spore germination, and germ tube elongation in a concentration-dependent manner (P<0.05). In vivo experiments showed that 4000 mg L−1 vanillin treatment inhibited cherry tomato gray mold and black rot occurrence. Besides, intercellular electrolytes, soluble proteins, and soluble sugars leakage indicated that 50 or 100 mg L−1 vanillin treatment increased Botrytis cinerea and Alternaria alternata membrane permeability. The increase of malondialdehyde and hydrogen peroxide contents confirmed that 50 or 100 mg L−1 vanillin treatment damages the pathogen membranes. Importantly, vanillin treatment inhibited the pathogenicity-related enzyme activities of the two pathogens to reduce their infection ability, among them PL enzyme activity in A. alternata was most inhibited, reducing by 94.7 % at 6 h treated with 100 mg L-1 vanillin. The hyphae morphology of the two pathogens changed, the mycelia were severely damaged, and the hyphae surface was deformed, shrunk, or even broken after 100 mg L−1 vanillin treatment. In summary, vanillin had a substantial inhibitory effect on postharvest gray mold and black rot in cherry tomato fruit. Therefore, vanillin can be an effective alternative to prevent and control cherry tomato postharvest diseases

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

PurposeIsorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis.MethodsIn vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR.ResultsIn HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1β, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro.ConclusionsIsorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Design, Synthesis, and Fungicidal Activities of Novel Piperidyl Thiazole Derivatives Containing Oxime Ether or Oxime Ester Moieties.

To explore the influence of the positions of the two nitrogen atoms on the thiazole ring and the isoxazoline ring on the activity, a series of novel piperidyl thiazole derivatives containing oxime ether and oxime ester moieties with two nitrogen atoms on the same or opposite sides have been designed, synthesized, and first evaluated for their fungicidal activities against Phytophthora capsici in vitro. The bioassay results showed that the target compounds possessed moderate to good fungicidal activities against P. capsici, among which oxime ether compound 11b shows the highest fungicidal activity in vitro (EC50 = 0.0104 μg/mL) which is higher than dimethomorph (EC50 = 0.1148 μg/mL) and diacetylenyl amide (EC50 = 0.040 μg/mL). Compared with oxime ether compounds (the two nitrogen atoms are on the opposite sides), the activities of oxime ester compounds were significantly reduced. It is different from the commercial fungicide fluoxapiprolin, and the activities of the compounds with the two nitrogen atoms on the same side were significantly reduced compared to the compounds with the two nitrogen atoms on the opposite sides. Moreover, compounds 11b, 11d, 11e, and 11g showed moderate to good antifungal activities in vivo against Phytophthora capsici, Pseudoperonospora cubensis, and Phytophthora infestans. Scanning electron microscopy of compound 11b on the hyphae morphology showed that compound 11b might cause mycelial abnormalities of P. capsici.