Two ferrous iron transporter-like proteins independently participate in asexual development under iron limitation and virulence in Beauveria bassiana.

Reductive assimilation pathway involves ferric reductase and ferrous iron transporter, which is integral for fungal iron acquisition. A family of ferric reductase-like proteins has been functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. In this investigation, two ferrous iron transporter-like proteins (Ftr) were functionally annotated in B. bassiana. BbFtr1 and BbFtr2 displayed high similarity in structure and were associated with the plasma and nuclear membrane. Their losses had no negatively influence on fungal growth on various nutrients and development under the iron-replete condition. Single mutants of BbFTR1 and BbFTR2 displayed the iron-availability dependent developmental defects, and double mutant exhibited the significantly impaired developmental potential under the iron-limited conditions. In insect bioassay, the double mutant also showed the weaker virulence than either of two single disruption mutants. These results suggested that two ferrous iron transporter-like proteins function independently in fungal physiologies under the iron-deficient condition. Intriguingly, a bZIP transcription factor BbHapX was required for expression of BbFTR1 and BbFTR2 under iron-depleted conditions. This study enhances our understanding of the iron uptake system in the filamentous entomopathogenic fungi.

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm-1 attributed to carbon­deuterium bending and at 477 cm-1, attributed to the nitrogen­deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

Physiological response of chestnuts (Castanea mollissima Blume) infected by pathogenic fungi and their correlation with fruit decay.

The postharvest decay of chestnuts (Castanea mollissima Blume) limits their industrial utilization, and pathogenic fungi are the main cause for chestnut decay. In this study, the physiological changes and their correlation with the rotting degree of chestnuts were investigated during single or mixed infection by Fusarium proliferatum, Penicillium crustosum, and Alternaria alternata. During the infection period, the activities of cell wall degrading enzymes (CWDEs) and antioxidant enzymes firstly increased and then decreased, the contents of nutrients decreased, but the levels of physiological indexes increased. The mycelium and spores of pathogenic fungi colonized the intercellular space, and then the mycelium covered the entire tissue surface of chestnuts, leading to the disappearance of cell structure. Notably, the most prominent changes in physiological indexes and ultrastructure were observed in chestnuts infected with three pathogenic fungi. Furthermore, the rotting degree of chestnuts was positively correlated with the levels of CWDEs, antioxidant enzymes and malondialdehyde.

Lipopeptide C17 Fengycin B Exhibits a Novel Antifungal Mechanism by Triggering Metacaspase-Dependent Apoptosis in Fusarium oxysporum.

Fusarium wilt is a worldwide soil-borne fungal disease caused by Fusarium oxysporum that causes serious damage to agricultural products. Therefore, preventing and treating fusarium wilt is of great significance. In this study, we purified ten single lipopeptide fengycin components from Bacillus subtilis FAJT-4 and found that C17 fengycin B inhibited the growth of F. oxysporum FJAT-31362. We observed early apoptosis hallmarks, including reactive oxygen species accumulation, mitochondrial dysfunction, and phosphatidylserine externalization in C17 fengycin B-treated F. oxysporum cells. Further data showed that C17 fengycin B induces cell apoptosis in a metacaspase-dependent manner. Importantly, we found that the expression of autophagy-related genes in the TOR signaling pathway was significantly upregulated; simultaneously, the accumulation of acidic autophagy vacuoles in F. oxysporum cell indicated that the autophagy pathway was activated during apoptosis induced by C17 fengycin B. Therefore, this study provides new insights into the antifungal mechanism of fengycin.

Lipopeptides from Bacillus velezensis induced apoptosis-like cell death in the pathogenic fungus Fusarium concentricum.

AIMS: Stem rot caused by Fusarium concentricum is a new disease of Paris polyphylla reported by our research group. The present study investigates the growth inhibitory and apoptotic effects of Bacillus velezensis FJAT-54560 lipopeptide against F. concentricum. METHODS AND RESULTS: HPLC preparation and LC-MS analysis results show that the crude lipopeptides secreted by Bacillus velezensis FJAT-54560 isolated from Jasminum sambac consist of C14-17 iturin A, C14 fengycin B, C16 fengycin A/A2, C18 fengycin A, C20 fengycin B2, C21 fengycin A2, C22-23 fengycin A, C12-16 surfactin A, and C15 surfactin A derivatives. The mass ratios (g/g) of iturin, fengycin, and surfactin in lipopeptides are 2.40, 67.51, and 30.08%, respectively. Through inhibition zone and inhibition rate experiments, we found that crude lipopeptides and purified fengycin exhibit strong antifungal activity against F. concentricum, including accumulation of reactive oxygen species, loss of mitochondrial membrane potential, DNA fragmentation, Ca2+ accumulation, chromatin condensation, and phosphatidylserine externalization. Transcriptomic analysis indicates that crude lipopeptide-induced apoptosis in F. concentricum cells may be mediated by apoptosis-inducing factors and apoptosis mediators and can serve as a metacaspase-independent model. CONCLUSION: Lipopeptides from Bacillus velezensis FJAT-54560 can control the pathogenic fungus F. concentricum by inducing apoptosis.

Sulfonate Derivatives Containing a Kakuol Moiety as Potential Fungicidal Candidates: Design, Synthesis and Antifungal Activity Evaluation.

As a part of our continuing exploration to discover new potential promising fungicide candidates, eighteen sulfonate derivatives (3a-3r) containing a kakuol moiety were designed and synthesized. Synthetic sulfonate derivatives were tested comprehensively for antifungal activities against four plant pathogenic fungi (Botrytis (B.) cinerea, Valsa (V.) mali, Fusarium (F.) graminearum, Sclerotinia (S.) sclerotiorum), and their structure activity relationships were summarized. Especially, derivatives 3i and 3j exhibited remarkable activity against V. mali, with the inhibition rates of 99.8 and 100%, which were slightly superior to that of carbendazim (98.9%), a reference fungicide. Moreover, derivatives 3a, 3k and 3q possess the broader antifungal spectrum against three tested plant pathogenic fungi with inhibition rates over 60%. Structure-activity relationship (SAR) analysis indicated that the introduction of 2-F or 3-F into the benzene ring would give rise to a remarkable increase of the antifungal activity against V. mali.

Rearing zombie flies: Laboratory culturing of the behaviourally manipulating fungal pathogen Entomophthora muscae.

Insect pathogenic fungi (IPF) and insects have ubiquitous interactions in nature. The extent of these interkingdom host-pathogen interactions are both complex and diverse. Some IPF, notably of the order Entomophthorales, manipulate their species-specific host before death. The fungus-induced altered insect behaviours are sequential and can accurately be repeatedly characterised temporally, making them a valuable model for understanding the molecular and chemical underpinnings of behaviour and host-pathogen co-evolutionary biology. Here, we present methods for the isolation and laboratory culturing of the emerging behaviourally manipulating model IPF Entomophthora muscae for experimentation.•E. muscae isolation and culturing in vitro.•Establishing and maintaining an E. muscae culture in vivo in houseflies (Musca domestica).•Controlled E. muscae infections for virulence experiments and quantification of conidia discharge per cadaver.

On-site detection of Sporothrix globosa using immunoglobulin Y, magnetic separation and loop-mediated isothermal amplification.

BACKGROUND: Sporothrix globosa (S. globosa) is an agricultural activity-related but neglected pathogenic fungus responsible for sporotrichosis. Timely detection is crucial for managing and preventing its spread. However, due to the lack of efficient recognition elements for enriching S. globosa, the current approaches for detecting S. globosa are not simple and/or sensitive enough. This hinders their wider application of fast screening. RESULTS: Herein, we successfully prepared immunoglobulin Y (IgY) targeting S. globosa, and developed a rapid and accurate detection method, improving upon current limited and inadequate detection approaches. Our method combined the use of IgY and loop-mediated isothermal amplification (LAMP) to enhance detection sensitivity and specificity simultaneously. The IgY was fabricated on magnetic beads to specifically concentrate S. globosa in samples, while LAMP amplified the captured target after simple boiling DNA extraction. By using our method, as low as 4.66 × 102 Cells mL-1S. globosa was accurately detected in soil and corn straw samples. We further integrated this assay into a portable toolbox for sample-to-result detection in resource-limited areas. By using this toolbox, we have colorimetrically detected soil and corn straw samples contaminated by S. globosa, suggesting the promising on-site detection potential. SIGNIFICANCE AND NOVELTY: A new IgY recognizing S. globosa was prepared. Through the combination of IgY enrichment and LAMP amplification, the detection sensitivity and specificity were improved simultaneously. This method eliminated thermal cycling, simplified the operation, and reduced the analysis time. Compared to existing methods, our approach is more suitable for on-site detection and can significantly improve public health responses to sporotrichosis outbreaks.

Three previously undescribed metabolites from Cordyceps cicadae JXCH-1, an entomopathogenic fungus.

Three previously undescribed compounds, cordycicadione (1), cordycicadin F (2), and 7-hydroxybassiatin (3), were isolated from the cultures of Cordyceps cicadae JXCH1, an entomopathogenic fungus. Their structures and relative configurations were elucidated primarily by NMR spectroscopic analysis. The absolute configurations of 1 and 2 were determined by ECD calculations. Single-crystal X-ray diffraction method was adopted to determine the absolute configuration of 3. Compound 2 is a polycyclic polyketide with an unusual enol ether moiety and a spiro ring. The compounds obtained in this study were subjected to screening their inhibition against the proliferation of the human lung cancer cell line A549 and the production of nitric oxide in murine macrophages RAW264.7.

An assembled bacterial community associated with Artemisia annua L. causes plant protection against a pathogenic fungus.

The microorganisms associated with a plant influence its growth and fitness. These microorganisms accumulate on the aerial and root surfaces of plants, as well as within the plants, as endophytes, although how the interaction between microorganisms protects the plant from pathogens is still little understood. In the current study, the impact of assembled the bacterial communities against the pathogenic fungus to promote Artemisia annua L. growths was investigated. We established a model of bacterium-fungus-plant system. Eight bacterial strains and a fungal pathogen Globisporangium ultimum (Glo) were isolated from wild A. annua roots and leaves, respectively. We assembled the six-bacteria community (C6: Rhizobium pusense, Paracoccus sp., Flavobacterium sp., Brevundimonas sp., Stenotrophomonas sp., and Bacillus sp.) with inhibition, and eight-bacteria community (C8) composing of C6 plus another two bacteria (Brevibacillus nitrificans and Cupriavidus sp.) without inhibition against Glo in individually dual culture assays. Inoculation of seedlings with C8 significantly reduced impact of Glo. The growth and disease suppression of A. annua seedlings inoculated with C8 + Glo were significantly better than those of seedlings inoculated with only Glo. C8 had more inhibitory effects on Glo, and also enhanced the contents of four metabolites in seedling roots compared to Glo treatment only. Additionally, the inhibitory effects of root extracts from A. annua seedlings showed that Glo was most sensitive, the degree of eight bacteria sensitivity were various with different concentrations. Our findings suggested that the non-inhibitory bacteria played a vital role in the bacterial community composition and that some bacterial taxa were associated with disease suppression. The construction of a defined assembled bacterial community could be used as a biological fungicide, promoting biological disease control of plants.

Effects of continuous cropping on fungal community diversity and soil metabolites in soybean roots.

IMPORTANCE: Soybean yield can be affected by soybean soil fungal communities in different tillage patterns. Soybean is an important food crop with great significance worldwide. Continuous cultivation resulted in soil nutrient deficiencies, disordered metabolism of root exudates, fungal pathogen accumulation, and an altered microbial community, which brought a drop in soybean output. In this study, taking the soybean agroecosystem in northeast China, we revealed the microbial ecology and soil metabolites spectrum, especially the diversity and composition of soil fungi and the correlation of pathogenic fungi, and discussed the mechanisms and the measures of alleviating the obstacles.

Whole-genome sequencing and functional annotation of pathogenic Paraconiothyrium brasiliense causing human cellulitis.

BACKGROUND: A pathogenic filamentous fungus causing eyelid cellulitis was isolated from the secretion from a patient's left eyelid, and a phylogenetic analysis based on the rDNA internal transcribed spacer region (ITS) and single-copy gene families identified the isolated strain as Paraconiothyrium brasiliense. The genus Paraconiothyrium contains the major plant pathogenic fungi, and in our study, P. brasiliense was identified for the first time as causing human infection. To comprehensively analyze the pathogenicity, and proteomics of the isolated strain from a genetic perspective, whole-genome sequencing was performed with the Illumina NovaSeq and Oxford Nanopore Technologies platforms, and a bioinformatics analysis was performed with BLAST against genome sequences in various publicly available databases. RESULTS: The genome of P. brasiliense GGX 413 is 39.49 Mb in length, with a 51.2% GC content, and encodes 13,057 protein-coding genes and 181 noncoding RNAs. Functional annotation showed that 592 genes encode virulence factors that are involved in human disease, including 61 lethal virulence factors and 30 hypervirulence factors. Fifty-four of these 592 virulence genes are related to carbohydrate-active enzymes, including 46 genes encoding secretory CAZymes, and 119 associated with peptidases, including 70 genes encoding secretory peptidases, and 27 are involved in secondary metabolite synthesis, including four that are associated with terpenoid metabolism. CONCLUSIONS: This study establishes the genomic resources of P. brasiliense and provides a theoretical basis for future studies of the pathogenic mechanism of its infection of humans, the treatment of the diseases caused, and related research.

Efficient multiple gene knockout in Colletotrichum higginsianum via CRISPR/Cas9 ribonucleoprotein and URA3-based marker recycling.

Colletotrichum higginsianum is a hemibiotrophic pathogen that causes anthracnose disease on crucifer hosts, including Arabidopsis thaliana. Despite the availability of genomic and transcriptomic information and the ability to transform both organisms, identifying C. higginsianum genes involved in virulence has been challenging due to recalcitrance to gene targeting and redundancy of virulence factors. To overcome these obstacles, we developed an efficient method for multiple gene disruption in C. higginsianum by combining CRISPR/Cas9 and a URA3-based marker recycling system. Our method significantly increased the efficiency of gene knockout via homologous recombination by introducing genomic DNA double-strand breaks. We demonstrated the applicability of the URA3-based marker recycling system for multiple gene targeting in the same strain. Using our technology, we successfully targeted two melanin biosynthesis genes, SCD1 and PKS1, which resulted in deficiency in melanization and loss of pathogenicity in the mutants. Our findings demonstrate the effectiveness of our methods in analysing virulence factors in C. higginsianum, thus accelerating research on plant-fungus interactions.

Natural products from marine fungi as a source against agricultural pathogenic fungi.

There are many kinds of agricultural pathogenic fungi, which may belong to pathogenic fungi in different species, such as Fusarium, Alternaria, Colletotrichum, Phytophthora, and other agricultural pathogens. Pathogenic fungi from different sources are widely distributed in agriculture, which threaten the lives of crops around the world and caused great damage to agricultural production and economic benefits. Due to the particularity of the marine environment, marine-derived fungi could produce natural compounds with unique structures, rich diversities, and significant bioactivities. Since marine natural products with different structural characteristics could inhibit different kinds of agricultural pathogenic fungi, secondary metabolites with antifungal activity could be used as lead compounds against agricultural pathogenic fungi. In order to summarize the structural characteristics of marine natural products against agricultural pathogenic fungi, this review systematically overview the activities against agricultural pathogenic fungi of 198 secondary metabolites from different marine fungal sources. A total of 92 references published from 1998 to 2022 were cited. KEY POINTS: • Pathogenic fungi, which could cause damage to agriculture, were classified. • Structurally diverse antifungal compounds from marine-derived fungi were summarized. • The sources and distributions of these bioactive metabolites were analyzed.

First report of Sclerotinia sclerotiorum causing stem canker on industrial hemp in South Carolina, USA.

On Jun. 20th, 2022, thirty industrial hemp (Cannabis sativa L.) plants (cv. Peach Haze) were vegetatively propagated, grown in a greenhouse for 21 days, and transplanted to a field at The Hemp Mine located in Fair Play, SC. Near the time of harvest (Nov. 17th, 2022), significant mycelial growth was noticed within the floral structure on 30% of plants. Three diseased plants were submitted to the Clemson University Plant and Pest Diagnostic Clinic. Stem cankers were observed on all three plants. Sclerotia typical of Sclerotinia spp. were found inside the stems of two plants. Two pure isolates were obtained by placing a sclerotium from each plant onto an acidified potato dextrose agar (APDA) plate and transferring a hyphal tip to a new APDA plate. After a 7-day-long growth at 25°C under a 24-h photoperiod, both isolates (22-1002-A and B) produced white and sparse mycelia and dark brownish to blackish sclerotia typical of S. sclerotiorum (aver. 36.5 per 90-mm plate). Sclerotia (n=50) were spherical (46%), oval (46%), or irregular (8%) and measured 1.8 to 7.2 × 1.6 to 4.5 mm (aver. 3.6 ± 1.2 × 2.7 ± 0.6 mm). No spores were produced. Sequences of the internal transcribed spacer region including the 5.8S ribosomal RNA gene (GenBank accession no. OQ749889) and the glyceraldehyde 3-phosphate dehydrogenases (G3PDH) gene (OQ790148) of 22-1002-A are 99.8% and 100% identical to those of a S. sclerotiorum isolate LAS01 on industrial hemp (MW079844 and MW082601; Garfinkel 2021). The G3PDH sequence of 22-1002-A is also 100% identical to that of ATCC 18683 (JQ036048), an authenticated S. sclerotiorum strain used for whole genome sequencing (Derbyshire et al. 2017). Ten healthy 'Peach Haze' plants (approx. 10 to 15" tall) grown in 6" pots were used in a pathogenicity test. The epidermis layer of each main stem was slightly wounded (2 × 2 mm2, 1 mm deep) using a sterile dissecting blade. A 5 × 5 mm2 mycelial plug of 22-1002-A was placed onto the wound of each of five plants, while APDA plugs were used for five control plants. Parafilm was used to secure mycelial and sterile agar plugs. All plants were maintained in an indoor controlled environment (25°C, >60% humidity, 24-h photoperiod). Stem cankers were visible on all inoculated plants 5 days after inoculation (DAI). Four of the five inoculated plants had noticeable yellowing and wilting on the foliage 9 DAI, while control plants remained asymptomatic. Elongated and tan-colored cankers (44.3 to 86.2 mm long, aver. 63.1 ± 18.3 mm) were developed at the wounded sites of inoculated plants. Wounded sites of control plants remained green in color and only slightly expanded in length (aver. 3.6 ± 0.8 mm). Tissue was excised from the canker margin of each inoculated plant and the wounded site of each control plant, surface sterilized with 10% bleach for 1 min, rinsed in sterile water, placed onto APDA, and incubated at 25°C. Sclerotia-producing colonies typical of S. sclerotiorum were recovered from all inoculated plants after 6 days, but not from any control plants. Sclerotinia sclerotiorum has a host range of more than 400 plant species (Boland and Hall 1994). This fungus causing stem canker on industrial hemp were reported from MT (Shaw 1973) and OR (Garfinkel 2021) in the USA and Canada (Bains et al. 2000). This is the first report of this disease in SC. Industrial hemp is an emerging crop in SC. The detection of this disease helps SC growers to take actions to monitor and prevent disease outbreak as well as develop an effective management practice when it occurs.

Chromosome-level analysis of the Colletotrichum graminicola genome reveals the unique characteristics of core and minichromosomes.

The fungal pathogen Colletotrichum graminicola causes the anthracnose of maize (Zea mays) and is responsible for significant yield losses worldwide. The genome of C. graminicola was sequenced in 2012 using Sanger sequencing, 454 pyrosequencing, and an optical map to obtain an assembly of 13 pseudochromosomes. We re-sequenced the genome using a combination of short-read (Illumina) and long-read (PacBio) technologies to obtain a chromosome-level assembly. The new version of the genome sequence has 13 chromosomes with a total length of 57.43 Mb. We detected 66 (23.62 Mb) structural rearrangements in the new assembly with respect to the previous version, consisting of 61 (21.98 Mb) translocations, 1 (1.41 Mb) inversion, and 4 (221 Kb) duplications. We annotated the genome and obtained 15,118 predicted genes and 3,614 new gene models compared to the previous version of the assembly. We show that 25.88% of the new assembly is composed of repetitive DNA elements (13.68% more than the previous assembly version), which are mostly found in gene-sparse regions. We describe genomic compartmentalization consisting of repeat-rich and gene-poor regions vs. repeat-poor and gene-rich regions. A total of 1,140 secreted proteins were found mainly in repeat-rich regions. We also found that ~75% of the three smallest chromosomes (minichromosomes, between 730 and 551 Kb) are strongly affected by repeat-induced point mutation (RIP) compared with 28% of the larger chromosomes. The gene content of the minichromosomes (MCs) comprises 121 genes, of which 83.6% are hypothetical proteins with no predicted function, while the mean percentage of Chr1-Chr10 is 36.5%. No predicted secreted proteins are present in the MCs. Interestingly, only 2% of the genes in Chr11 have homologs in other strains of C. graminicola, while Chr12 and 13 have 58 and 57%, respectively, raising the question as to whether Chrs12 and 13 are dispensable. The core chromosomes (Chr1-Chr10) are very different with respect to the MCs (Chr11-Chr13) in terms of the content and sequence features. We hypothesize that the higher density of repetitive elements and RIPs in the MCs may be linked to the adaptation and/or host co-evolution of this pathogenic fungus.

Soil cadmium stress affects the phyllosphere microbiome and associated pathogen resistance differently in male and female poplars.

Microorganisms associated with the phyllosphere play a crucial role in protecting plants from diseases, and their composition and diversity are strongly influenced by heavy metal contaminants. Dioecious plants are known to exhibit sexual dimorphism in metal accumulation and tolerance between male and female individuals. Hence, in this study we used male and female full-siblings of Populus deltoides to investigate whether the two sexes present differences in their phyllosphere microbiome structures and in their associated resistance to the leaf pathogenic fungus Pestalotiopsis microspora after exposure to excess soil cadmium (Cd). We found that Cd-treated male plants grew better and accumulated more leaf Cd than females. Cd stress reduced the lesion areas on leaves of both sexes after pathogen infection, but male plants exhibited better resistance than females. More importantly, Cd exposure differentially altered the structure and function of the phyllosphere microbiomes between the male and female plants, with more abundant ecologically beneficial microbes and decreased pathogenic fungal taxa harbored by male plants. In vitro toxicity tests suggested that the sexual difference in pathogen resistance could be attribute to both direct Cd toxicity and indirect shifts in the phyllosphere microbiome. This study provides new information relevant for understanding the underlying mechanisms of the effects of heavy metals involved in plant-pathogen interactions.

Whole genome sequence of Cryptosphaeria pullmanensis, an important pathogenic fungus potentially threatening crop and forestry production.

Many fungal members of the Diatrypaceae family are pathogenic towards plants and are widely distributed globally. Cryptosphaeria pullmanensis is a pathogenic fungus that infects populus and walnut trees, causing their death. We sequenced the genome of C. pullmanensis based on a combination of Nanopore PromethION and Illumina NovaSeq PE150 platforms, and functionally annotated the sequences using a number of open-access databases. This is the first report of the genome-scale assembly and annotation for C. pullmanensis, the first species of the genus Cryptosphaeria to be sequenced. We obtained 13 contigs with an N50 contig size of 7,095,780 bp, a GC content ratio of 43.23% and a genome size of 56.72 Mb with 10,474 putative coding genes. Comparative genomic analysis against the genomes of seven Ascomycetes fungal strains was performed. Among the seven species tested, the Eutypa lata genome displayed the highest similarity to the C. pullmanensis genome in terms of collinearity and homologous gene content. This study has provided a genetic resource that offers extensive information and a framework for future investigations into the transcriptome, proteome, and metabonome of C. pullmanensis to understand its molecular pathogenesis.