Major Fungal Pathogen Research Articles

Nano-pesticidal potential of Cassia fistula (L.) leaf synthesized silver nanoparticles (Ag@CfL-NPs): Deciphering the phytopathogenic inhibition and growth augmentation in Solanum lycopersicum (L.).

Plant-based synthesis of silver nanoparticles (Ag-NPs) has emerged as a potential alternative to traditional chemical synthesis methods. In this context, the aim of the present study was to synthesize Ag-NPs from Cassia fistula (L.) leaf extract and to evaluate their nano-pesticidal potential against major phyto-pathogens of tomato. From the data, it was found that particle size of spherical C. fistula leaf synthesized (Ag@CfL-NPs) varied from 10 to 20 nm, with the average diameter of 16 nm. Ag@CfL-NPs were validated and characterized by UV-visible spectroscopy (surface resonance peak λmax = 430 nm), energy dispersive spectrophotometer (EDX), Fourier transform infrared (FTIR), and X-ray diffraction pattern (XRD), and electron microscopy; scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The FTIR spectra verified the participation of various living molecules (aromatic/aliphatic moieties and proteins) in synthesized Ag@CfL-NPs. The anti-phytopathogenic potential of Ag@CfL-NPs was assessed under in vitro conditions. Increasing doses of Ag@CfL-NPs exhibited an inhibitory effect against bacterial pathogen Pseudomonas syringae and 400 μg Ag@CfL-NPs ml–1 caused a reduction in cellular viability, altered bacterial morphology, and caused cellular death Furthermore, Ag@CfL-NPs reduced exopolysaccharides (EPS) production and biofilm formation by P. syringae Additionally, Ag@CfL-NPs showed pronounced antifungal activity against major fungal pathogens. At 400 μg Ag@CfL-NPs ml–1, sensitivity of tested fungi followed the order: Fusarium oxysporum (76%) > R. solani (65%) > Sarocladium (39%). Furthermore, 400 μg Ag@CfL-NPs ml–1 inhibited the egg-hatching and increased larval mortality of Meloidogyne incognita by 82 and 65%, respectively, over control. Moreover, pot studies were performed to assess the efficacy of Ag@CfL-NPs to phyto-pathogens using tomato (Solanum lycopersicum L.) as a model crop. The applied phyto-pathogens suppressed the biological, physiological, and oxidative-stress responsiveness of tomatoes. However, 100 mg Ag@CfL-NPs kg–1 improved overall performance and dramatically increased the root length, dry biomass, total chlorophyll, carotenoid, peroxidase (POD), and phenylalanine ammonia lyase (PAL) activity over pathogens-challenged tomatoes. This study is anticipated to serve as an essential indication for synthesis of efficient nano-control agents, which would aid in the management of fatal phyto-pathogens causing significant losses to agricultural productivity. Overall, our findings imply that Ag@CfL-NPs as nano-pesticides might be used in green agriculture to manage the diseases and promote plant health in a sustainable way.

Prevalence and Antifungal Susceptibility of Clinically Relevant Candida Species, Identification of Candida auris and Kodamaea ohmeri in Bangladesh

Candida species are major fungal pathogens in humans. The aim of this study was to determine the prevalence of individual Candida species and their susceptibility to antifungal drugs among clinical isolates in a tertiary care hospital in Bangladesh. During a 10-month period in 2021, high vaginal swabs (HVSs), blood, and aural swabs were collected from 360 patients. From these specimens, Candida spp. was isolated from cultures on Sabouraud dextrose agar media, and phenotypic and genetic analyses were performed. A total of 109 isolates were recovered, and C. albicans accounted for 37%, being derived mostly from HVSs. Among non-albicans Candida (NAC), C. parapsilosis was the most frequent, followed by C. ciferrii, C. tropicalis, and C. glabrata. Three isolates from blood and two isolates from aural discharge were genetically identified as C. auris and Kodamaea ohmeri, respectively. NAC isolates were more resistant to fluconazole (overall rate, 29%) than C. albicans (10%). Candida isolates from blood showed 95% susceptibility to voriconazole and less susceptibility to fluconazole (67%). Two or three amino acid substitutions were detected in the ERG11 of two fluconazole-resistant C. albicans isolates. The present study is the first to reveal the prevalence of Candida species and their antifungal susceptibility in Bangladesh.

Metatranscriptomic Analysis Reveals Rich Mycoviral Diversity in Three Major Fungal Pathogens of Rice

In recent years, three major fungal diseases of rice, i.e., rice blast, rice false smut, and rice-sheath blight, have caused serious worldwide rice-yield reductions and are threatening global food security. Mycoviruses are ubiquitous in almost all major groups of filamentous fungi, oomycetes, and yeasts. To reveal the mycoviral diversity in three major fungal pathogens of rice, we performed a metatranscriptomic analysis of 343 strains, representing the three major fungal pathogens of rice, Pyricularia oryzae, Ustilaginoidea virens, and Rhizoctonia solani, sampled in southern China. The analysis identified 682 contigs representing the partial or complete genomes of 68 mycoviruses, with 42 described for the first time. These mycoviruses showed affinity with eight distinct lineages: Botourmiaviridae, Partitiviridae, Totiviridae, Chrysoviridae, Hypoviridae, Mitoviridae, Narnaviridae, and Polymycoviridae. More than half (36/68, 52.9%) of the viral sequences were predicted to be members of the families Narnaviridae and Botourmiaviridae. The members of the family Polymycoviridae were also identified for the first time in the three major fungal pathogens of rice. These findings are of great significance for understanding the diversity, origin, and evolution of, as well as the relationship between, genome structures and functions of mycoviruses in three major fungal pathogens of rice.

Chemoenzymatic Probes Reveal Peptidoglycan Recognition and Uptake Mechanisms in Candida albicans.

Candida albicans, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step in C. albicans pathogenesis and infections. However, due to the synthetic difficulties of obtaining peptidoglycan fragments for biological studies, mechanistic details of how C. albicans recognizes and uptakes these peptidoglycan fragments have not been well elucidated. Notably, previous works have solely focused on the synthetic peptidoglycan ligand, muramyl dipeptide (MDP), despite its poor hyphal-inducing activity in C. albicans. In this work, we isolated and purified natural peptidoglycan fragments via enzymatic degradation of bacteria cell wall sacculi and chemoenzymatically installed a series of functional d-amino acids into the natural muropeptide, creating peptidoglycan probes that bear photoaffinity, bio-orthogonal, or fluorescent functionality. Using these chemoenzymatic peptidoglycan probes, we established that natural peptidoglycan fragments, which are potent hyphal-inducers, interact with the C. albicans Cyr1 sensor protein in the in-gel fluorescence assay as well as in in vitro pulldown studies. Moreover, we established that bacterial peptidoglycan probes enter C. albicans cells via an energy-dependent endocytic process.

Rapid detection of 5 fungal diseases in sunflower (Helianthus annuus) using dual priming oligonucleotide system-based multiplex PCR and capillary electrophoresis.

Rapid and accurate diagnosis of fungal pathogens is essential for disease control in sunflower. In the present study, a multiplex PCR assay was developed based on the dual priming oligonucleotide (DPO) system, which was used to simultaneously detect and identify five major sunflower fungal pathogens. There was no cross-reactivity among the pathogens tested. In each reaction, 0.1 ng genomic DNA templates were sufficient to ensure specificity and accuracy. The system exhibited high adaptability over a wide range of annealing temperatures. No mismatch or nonspecific amplification was observed in the annealing temperatures tested. In combination with capillary electrophoresis, the DPO-primer-based multiplex PCR system provides a rapid, reliable and cost-efficient solution for the diagnosis of fungal pathogens in sunflower.

Repurposing of MitoTam: Novel Anti-Cancer Drug Candidate Exhibits Potent Activity against Major Protozoan and Fungal Pathogens.

ABSTRACTMany of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug candidate MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority.

Antifungal Potential of Three Plant Extracts in the Control of Some Major Fungi Associated with Cashew Seeds (Anacardium occidentale L.)

Cashew (Anacardium occidentale L.), is an economically export oriented crop that could plays an important role in the Cameroonian economy. Owing to its importance, a study was carried out to evaluate the potential antifungal activities of three plants extracts against three major fungal pathogens associated to its seeds. To achieve this objective, cashew seeds samples were collected from Cameroon and Chad, and fungal pathogens isolated on Potato Dextrose Agar (PDA) culture medium. In vitro antifungal activities of Callistemon viminalis, Cupressus lusitanica and Lantana camara were assessed on three major fungi; Cercospora sp., Colletotrichum gloeosporioides and Pestalotia heterocornis. Results showed that cashew seeds harbour a diversity of fungal species; the most frequent are Cercospora sp. (26%), followed by Aspergillus niger (17.78%) and P. heterocornis (15.6%). Aqueous extracts at 28 mg/ml and ethanolic extracts at 16 mg/ml of C. lusitanica and L. camara, inhibited at 100%, the radial growth and sporulation of all tested fungi. This preliminary work has opened up a possibility of the use of these extracts in the treatment of seed-borne fungi of cashew nuts. However further studies are still on-going under in vivo field conditions to practically evaluate on their potential in cashew seeds disease management.

A novel maize microRNA negatively regulates resistance to Fusarium verticillioides.

Although microRNAs (miRNAs) regulate the defence response against multiple pathogenic fungi in diverse plant species, few efforts have been devoted to deciphering the involvement of miRNA in resistance to Fusarium verticillioides, a major pathogenic fungus affecting maize production. In this study, we discovered a novel F. verticillioides‐responsive miRNA designated zma‐unmiR4 in maize kernels. The expression of zma‐unmiR4 was significantly repressed in the resistant maize line but induced in the susceptible lines upon exposure to F. verticillioides exposure, whereas its target gene ZmGA2ox4 exhibited the opposite pattern of expression. Heterologous overexpression of zma‐unmiR4 in Arabidopsis resulted in enhanced growth and compromised resistance to F. verticillioides. By contrast, transgenic plants overexpressing ZmGA2ox4 or the homologue AtGA2ox7 showed impaired growth and enhanced resistance to F. verticillioides. Moreover, zma‐unmiR4‐mediated suppression of AtGA2ox7 disturbed the accumulation of bioactive gibberellin (GA) in transgenic plants and perturbed the expression of a set of defence‐related genes in response to F. verticillioides. Exogenous application of GA or a GA biosynthesis inhibitor modulated F. verticillioides resistance in different plants. Taken together, our results suggest that the zma‐unmiR4–ZmGA2ox4 module might act as a major player in balancing growth and resistance to F. verticillioides in maize.

SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum

Stem rot, caused by Sclerotinia sclerotiorum has emerged as one of the major fungal pathogens of oilseed Brassica across the world. The pathogenic development is exquisitely dependent on reactive oxygen species (ROS) modulation. Cox17 is a crucial factor that shuttles copper ions from the cytosol to the mitochondria for the cytochrome c oxidase (CCO) assembly. Currently, no data is available regarding the impact of Cox17 in fungal pathogenesis. The present research was carried out to functionally characterize the role of Cox17 in S. sclerotiorum pathogenesis. SsCox17 transcripts showed high expression levels during inoculation on rapeseed. Intramitochondrial copper content and CCO activity were decreased in SsCox17 gene-silenced strains. The SsCox17 gene expression was up-regulated in the hyphae under oxidative stress and a deficiency response to oxidative stress was detected in SsCox17 gene-silenced strains. Compared to the S. sclerotiorum wild-type strain, there was a concomitant reduction in the virulence of SsCox17 gene-silenced strains. The SsCox17 overexpression strain was further found to increase copper content, CCO activity, tolerance to oxidative stress and virulence. We also observed a certain correlation of appressoria formation and SsCox17. These results provide evidence that SsCox17 is positively associated with fungal virulence and oxidative detoxification.

Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts

Plant-pathogenic fungi form intimate interactions with their associated bacterial microbiota during their entire life cycle. However, little is known about the structure, functions and interaction mechanisms of bacterial communities associated with fungal fruiting bodies (perithecia). Here we examined the bacterial microbiome of perithecia formed by Fusarium graminearum, the major pathogenic fungus causing Fusarium head blight in cereals. A total of 111 shared bacterial taxa were identified in the microbiome of 65 perithecium samples collected from 13 geographic locations. Within a representative culture collection, 113 isolates exhibited antagonistic activity against F. graminearum, with Pantoea agglomerans ZJU23 being the most efficient in reducing fungal growth and infectivity. Herbicolin A was identified as the key antifungal compound secreted by ZJU23. Genetic and chemical approaches led to the discovery of its biosynthetic gene cluster. Herbicolin A showed potent in vitro and in planta efficacy towards various fungal pathogens and fungicide-resistant isolates, and exerted a fungus-specific mode of action by directly binding and disrupting ergosterol-containing lipid rafts. Furthermore, herbicolin A exhibited substantially higher activity (between 5- and 141-fold higher) against the human opportunistic fungal pathogens Aspergillus fumigatus and Candida albicans in comparison with the clinically used fungicides amphotericin B and fluconazole. Its mode of action, which is distinct from that of other antifungal drugs, and its efficacy make herbicolin A a promising antifungal drug to combat devastating fungal pathogens, both in agricultural and clinical settings.

Genetic Diversity and Population Structure of Fusarium commune Causing Strawberry Root Rot in Southcentral China

Strawberry plants and fruits are vulnerable to infections by a broad range of pathogens and pests. However, knowledge about the epidemiology of pathogens causing strawberry diseases is limited. In this study, we analyzed Fusarium commune, a major fungal pathogen causing strawberry root rot, from diseased strawberry root tissues in southcentral China. A total of 354 isolates were obtained from 11 locations that spanned about 700 km from both south to north and east to west. Multilocus genotypes of all isolates were obtained using seven polymorphic simple sequence repeat markers developed in this study. Our analyses revealed significant genetic diversity within each of the 11 local populations of F. commune. STRUCTURE analysis revealed that the optimal number of genetic populations for the 354 strains was two, with most local geographic populations containing isolates in both genetic clusters. Interestingly, many isolates showed allelic ancestry to both genetic clusters, consistent with recent hybridization between the two genetic clusters. In addition, though alleles and genotypes were frequently shared among local populations, statistically significant genetic differentiations were found among the local populations. However, the observed F. commune population genetic distances were not correlated with geographic distances. Together, our analyses suggest that populations of F. commune causing strawberry root rot are likely endemic to southcentral China, with each local population containing shared and unique genetic elements. Though the observed gene flow among geographic regions was relatively low, human activities will likely accelerate pathogen dispersals, resulting in the generation of new genotypes through mating and recombination.

Blocking Polyphosphate Mobilization Inhibits Pho4 Activation and Virulence in the Pathogen Candida albicans.

ABSTRACTThe ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate is regulated by the Pho4 transcription factor and is important for both virulence and resistance to host-encountered stresses. All cells store phosphate in the form of polyphosphate (polyP), a ubiquitous polymer comprising tens to hundreds of phosphate residues. Release of phosphate from polyP is one of the first responses evoked in response to phosphate starvation, and here, we sought to explore the importance of polyP mobilization in the pathobiology of C. albicans. We found that two polyphosphatases, Ppn1 and Ppx1, function redundantly to release phosphate from polyP in C. albicans. Strikingly, we reveal that blocking polyP mobilization prevents the activation of the Pho4 transcription factor: following Pi starvation, Pho4 fails to accumulate in the nucleus and induce Pi acquisition genes in ppn1Δ ppx1Δ cells. Consequently, ppn1Δ ppx1Δ cells display impaired resistance to the same range of stresses that require Pho4 for survival. In addition, cells lacking both polyphosphatases are exquisitely sensitive to DNA replication stress, indicating that polyP mobilization is needed to support the phosphate-demanding process of DNA replication. Blocking polyP mobilization also results in significant morphological defects, as ppn1Δ ppx1Δ cells form large pseudohypha-like cells that are resistant to serum-induced hypha formation. Thus, polyP mobilization impacts key processes important for the pathobiology of C. albicans, and consistent with this, we found that blocking this process attenuates the virulence of this important human fungal pathogen.

Differential Functions of Individual Transcription Factor Binding Sites in the Tandem Repeats Found in Clinically Relevant cyp51A Promoters in Aspergillus fumigatus.

ABSTRACTAspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use, but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs, and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34- or 46-bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34 and TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element [SRE]) and AtrR binding sites (AtrR response element [ATRE]) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34-bp repeat of the TR34 promoter (further 5′ from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34- or 46-bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34- or 46-bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications.

Antibacterial Activity of Copper Nanoparticles against Xanthomonas campestris pv. vesicatoria in Tomato Plants.

Copper-based bactericides have appeared as a new tool in crop protection and offer an effective solution to combat bacterial resistance. In this work, two copper nanoparticle products that were previously synthesized and evaluated against major bacterial and fungal pathogens were tested on their ability to control the bacterial spot disease of tomato. Growth of Xanthomonas campestris pv. vesicatoria, the causal agent of the disease, was significantly suppressed by both nanoparticles, which had superior function compared to conventional commercial formulations of copper. X-ray fluorescence spectrometry measurements in tomato leaves revealed that bioavailability of copper is superior in the case of nanoparticles compared to conventional formulations and is dependent on synthesis rather than size. This is the first report correlating bioavailability of copper to nanoparticle efficacy.

The population genetics of adaptation through copy number variation in a fungal plant pathogen.

Microbial pathogens can adapt rapidly to changing environments such as the application of pesticides or host resistance. Copy number variations (CNVs) are a major source of adaptive genetic variation for recent adaptation. Here, we analyse how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to the host environment and fungicide applications. We screen the genomes of 125 isolates sampled across a worldwide set of populations and identify a total of 7,879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. Although CNVs are generally under negative selection, we find that genes affected by CNVs are enriched in functions related to host exploitation (i.e., effectors and cell-wall-degrading enzymes). We perform genome-wide association studies (GWAS) and identify a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance and a duplication encompassing an effector gene affecting virulence. We show that the adaptive CNVs were probably created by recently active transposable element families. Moreover, we find that specific transposable element families are important drivers of recent gene CNV. Finally, we use a genome-wide single nucleotide polymorphism data set to replicate the GWAS and contrast it with the CNV-focused analysis. Together, our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen.

Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.

ABSTRACTCryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the “AS15 sequence” was effective at high concentrations. A modified peptide, “AW9-Ma” showed a MIC of 64 μg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 μg/mL, the same as the cdc50Δ mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4-ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity.IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.

Potential antifungal impact of citral and linalool administered individually or combined with fluconazole against clinical isolates of Candida krusei

Introduction: Candida krusei is recognized as a major fungal pathogen in patients with immunodeficiency disorders. The present study aimed at investigating the anticandidal activities of citral and linalool combined with fluconazole (FLZ) against FLZ-resistant C. krusei strains. Methods: Antifungal activities were evaluated by the broth microdilution (MD) method to determine the minimum inhibitory and fungicidal concentrations (namely, MICs and MFCs) according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document. The interactions were further evaluated using fractional inhibitory concentration indices (FICIs) for combinations of citral+FLZ and linalool+FLZ, calculated from checkerboard MD assays. Results: The mean ± standard deviation (SD) MIC values of citral, linalool, and FLZ against the C. krusei isolates were 70.23 ± 17, 150 ± 38.73, and 74.66 ± 36.95 μg/mL, respectively. Some fungicidal activities were also observed for citral (2.5) and linalool (1.53) against the C. krusei isolates. The FICI values of citral+FLZ and linalool+FLZ for the C. krusei isolates ranged from 0.4 to 1.00 and 0.19 to 0.63, respectively. The additive and synergistic interactions of linalool + FLZ were further observed in 12 (57.1%) and 9 (42.9%) C. krusei isolates. However, there was an additive interaction for citral + FLZ in 17 (80.9%) isolates. They also showed a synergistic interaction in only four (19.1%) isolates. Moreover, linalool and citral plus FLZ did not have any antagonistic effect on any isolates. Conclusion: The study findings support the possible capabilities of citral and linalool, as anticandidal agents, and FLZ might be supplemented with citral and/or linalool for treating FLZ-resistant C. krusei infections.

Antifungal Mechanisms of a Chinese Herbal Medicine, Cao Huang Gui Xiang, Against Candida Species.

Cao Huang Gui Xiang (CHGX) formula, a Chinese herbal medicine, has been empirically used for the treatment of Candida infections. In the present study, we discovered that the CHGX showed potent antifungal activities against the major human fungal pathogen Candida albicans and other clinical Candida species. Besides, we indicated that CHGX had in vivo efficacy on treating C. albicans infection in mice without noticeable toxicity at the clinical therapeutic concentration. We then set out to investigate the antifungal mechanisms of CHGX against C. albicans. We found that CHGX played an important role in inhibiting biofilm formation and filament development, two critical virulence factors of C. albicans. We further demonstrated that CHGX disrupted cell membrane integrity, triggered the accumulation of reactive oxygen species (ROS) and consumption of adenosine triphosphate (ATP), followed by a rapid fungal cell death in C. albicans. Multiple pathways, including the conserved Ras1-cAMP pathway and mitochondrial protein Mcu1 are involved in CHGX-induced cell death. Our finding expands the understanding of antifungal mechanism of CHGX against C. albicans, and provides new insights in treating patients with Candida infections in clinical practice.

Efficacy of Bacillus subtilis XZ18-3 as a Biocontrol Agent against Rhizoctonia cerealis on Wheat

Rhizoctonia cerealis is a major fungal pathogen of wheat that causes great yield losses in all wheat-growing regions of the world. The biocontrol agent Bacillus subtilis XZ18-3 was investigated for inhibiting R. cerealis growth in wheat. The results of the mycelial growth test showed that the sterile filtrate of B. subtilis XZ18-3 could significantly inhibit the mycelial growth of R. cerealis and cause swelling and rupture of the mycelium. Observation by transmission electron microscopy indicated that the sterile filtrate could penetrate the cellular membrane of Rhizoctoniacerealis, resulting in organelle destruction. The effect of the sterile filtrates on the pathogen cells, shown through fluorescent microscopy using different stains, revealed the mechanism by which the sterile filtrate caused DNA fragmentation, accumulation of ROS and changes in cell membrane permeability. To reach a better treatment of the soil-borne fungi, the components of a wettable powder were screened and an optimised formula determined (30.0% kaolin, 4.0% polyvinyl alcohol, 8.0% Tween-80, 2.0% polyethylene glycol and 100% fermentation broth). A quality index analysis revealed that the wetting powder reached acceptable biological pesticide standards. Pot control experiments showed that the wettable powder of B. subtilis XZ18-3 effectively controlled the pathogens with an efficacy of 88.28%. This study has provided the potential biocontrol agents (BCAs) for wheat sharp eyespot disease.

Beijerinckia fluminensis BFC-33, a novel multi-stress-tolerant soil bacterium: Deciphering the stress amelioration, phytopathogenic inhibition and growth promotion in Triticum aestivum (L.)

Environmental challenges like drought, salinity, heavy metals and pesticides directly/indirectly influence the environment and decreased the agricultural output. During its long developmental stages, cereal crops including wheat is exposed to a variety of abiotic/biotic stressors. Certain beneficial soil bacteria that can ameliorate environmental stresses can be exploited as crop growth promoters/enhancers under adverse situations. In this study, Beijerinckia fluminensis BFC-33 (accession no. MT672580) isolated from potato rhizosphere tolerated variousabiotic (drought, salinity, temperature, heavy metals, and fungicides) stresses. Strain BFC-33 demonstrated multifarious plant-growth-promoting (PGP) characteristics, such as the production of indole-3-acetic acid, P-solubilization, ACC deaminase, ammonia, siderophore, HCN, EPS, and extracellular enzymes. The antagonistic potential of BFC-33 against major fungal pathogens was ranked: Alternaria alternata (79.2%)>Rhizoctonia solani (69%)>Fusarium oxysporum (23.5%)>Ustilaginoidea virens (17%). Furthermore, bacterization of wheat seeds witha multi-stress-tolerant strain revealed B. fluminensis as a plant growth enhancer and biocontrol agent. For instance, increase in root length (cm) in BFC-33 inoculated wheat exposed to abiotic and biotic stresses at the seedling stage was ranked: B. fluminensis (24.2)>B. fluminensis + 100μgTBZLmL−1 (21.3) = B. fluminensis + 2%PEG (21.3)>B. fluminensis + 100 mM NaCl (19.7)>B. fluminensis + 100μgPbmL−1 (19) = B. fluminensis 100μgMNZBmL−1 (19)>B. fluminensis + A. alternata (17.4)>B. fluminensis + 100μgCdmL−1 (17)>B. fluminensis + F. oxysporum (13.4). In addition, increase in carotenoid accumulation (mg g−1FW) in the foliage of BFC-33 inoculated wheat exposed to fungal infection was ranked: BFC-33 (3.88)>BFC-33+ A. alternata (3.0)>BFC-33+ R. solani (2.78)>BFC-33+ F. oxysporum (2.44). Moreover, BFC-33 inoculation significantly (p ≤ 0.05) reduced stress-induced stressor molecules (proline and TBARS) and electrolyte leakage. Furthermore, B. fluminensis BFC-33 potentially enhanced the defense responses in wheat seedlings by increasing phenylalanine ammonia lyase (PAL), β-1,3 glucanase, and polyphenol oxidase (PPO), which play a significant role in protecting plants from phytopathogens. Even so, by successfully establishing a product with the requisite effects under field settings, selecting multi-stress-tolerant and antagonistic plant growth promoting rhizobacteria (PGPRs) would be helpful to end-users. Future use of native multi-stress-tolerant bacteria as biocontrol agents in conjunction with existing drought, salinity, heavy metal, and pesticide tolerance might contribute to global food security.