Detection Of Fungal Pathogens Research Articles

Comparative Cross-Kingdom DDA- and DIA-PASEF Proteomic Profiling Reveals Novel Determinants of Fungal Virulence and a Putative Druggable Target.

Accurate and reliable detection of fungal pathogens presents an important hurdle to manage infections, especially considering that fungal pathogens, including the globally important human pathogen, Cryptococcus neoformans, have adapted diverse mechanisms to survive the hostile host environment and moderate virulence determinant production during coinfections. These pathogen adaptations present an opportunity for improvements (e.g., technological and computational) to better understand the interplay between a host and a pathogen during disease to uncover new strategies to overcome infection. In this study, we performed comparative proteomic profiling of an in vitro coinfection model across a range of fungal and bacterial burden loads in macrophages. Comparing data-dependent acquisition and data-independent acquisition enabled with parallel accumulation serial fragmentation technology, we quantified changes in dual-perspective proteome remodeling. We report enhanced and novel detection of pathogen proteins with data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF), especially for fungal proteins during single and dual infection of macrophages. Further characterization of a fungal protein detected only with DIA-PASEF uncovered a novel determinant of fungal virulence, including altered capsule and melanin production, thermotolerance, and macrophage infectivity, supporting proteomics advances for the discovery of a novel putative druggable target to suppress C. neoformans pathogenicity.

Molecular Detection of Dermatophytes and Nondermatophytes in Onychomycosis in Antalya, Turkey.

Onychomycosis is a chronic nail infection, and dermatophytes, yeasts, and nondermatophytic molds may be the causative agents. This study aimed to determine the etiological agents of onychomycosis by using conventional and molecular methods. Between June 2020 and July 2021, 37 patients with a presumptive diagnosis of onychomycosis and mycological evidence (culture and/or EUROArray Dermatomycosis assay) were included in the study. Organisms detected in cultured nail specimens were identified by combined phenotypic characteristics and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). An EUROarray Dermatomycosis assay was used for molecular detection of fungal pathogens. The EUROArray Dermatomycosis assay was positive for a single fungal target in 23 samples, and 14 samples were positive by culture. The most common pathogen was Trichophyton rubrum in both methods. Coinfection was detected in 14 samples by using molecular methods, and Trichophyton rubrum and Fusarium solani (9 samples) were the most common pathogens detected together. Trichophyton spp., nondermatophyte molds, and Candida spp. were detected in 33 (89.2%), 16 (43.2%), and 6 (16.2%) samples, respectively, when the two methods were evaluated together. Our results revealed that fungal culture allows the diagnosis of onychomycosis, but it is not as sensitive as the EUROArray Dermatomycosis test, especially in patients receiving antifungal therapy.

Cascaded amplifying circuit enables sensitive detection of fungal pathogens

The rapid and accurate detection of fungal pathogens is of utmost importance in the fields of healthcare, food safety, and environmental monitoring. In this study, we implemented a cascaded amplifying circuit in Saccharomyces cerevisiae to improve the G protein-coupled receptor (GPCR) mediated fungal detection. The GPCR signaling pathway was coupled with the galactose-regulated (GAL) system and a positive feedback loop was implemented to enhance the performance of yeast biosensor. We systematically compared four generations of biosensors for detecting the mating pheromone of Candida albicans, and the best biosensor exhibited the limit of detection (LOD) as low as 0.25 pM and the limit of quantification (LOQ) of 1 pM after 2 h incubation. Subsequently, we developed a betaxanthin-based colorimetric module for the easy visualization of signal outputs, and the resulting biosensors can give reliable naked-eye readouts. In summary, we demonstrated that cascaded amplifying circuits could substantially improve the engineered yeast biosensors with a better sensitivity and signal output magnitude, which will pave the way for their real-world applications in public health.

Detection of Pathogenic Fungi Causing Root and Crown Rot Diseases in Strawberry Fragaria ananassa Duch

A study was conducted in the Department of Plant Protection, College of Agriculture, University of Anbar, aiming to identify the major fungi causing root and crown rot disease in strawberries through isolation and diagnosis using both morphological and molecular methods. the results of isolation and diagnosis from the affected plant parts revealed the prevalence of root and crown rot disease in all sampled areas exhibiting symptoms and signs of the disease. Seventeen isolates were obtained belonging to the Fusarium species, eight isolates belonged to the fungus Rhizoctonia solani, and seven isolates belonged to the spp. Neopestalotiapsis The fungal species were identified based on their taxonomic characteristics. DNA extraction was successfully performed on eleven isolated fungi from the roots and crowns of strawberry plants in different isolation areas. Partial diagnosis was conducted using PCR technique targeting the intergenic spacer region using primers (ITS1-ITS4) to obtain amplicons of 650 base pairs (bp) in size. The amplicons were then separated on 2% agarose gel, and the nucleotide base sequences of these isolates were determined and deposited in the gene bank.

Rapid and sensitive detection of two fungal pathogens in soybeans using the recombinase polymerase amplification/CRISPR-Cas12a method for potential on-site disease diagnosis.

Diaporthe aspalathi and Diaporthe caulivora are two of the fungal pathogens causing soybean stem canker (SSC) in soybean, which is one of the most widespread diseases in soybean growing regions and can cause 100% loss of yield. Current methods for the detection of fungal pathogens, including morphological identification and molecular detection, are mostly limited by the need for professional laboratories and staff. To develop a detection method for potential on-site diagnosis for two of the fungal pathogens causing SSC, we designed a rapid assay combining recombinase polymerase amplification (RPA) and CRISPR-Cas12a-based diagnostics to specifically detect D. aspalathi and D. caulivora. The translation elongation factor 1-alpha gene was employed as the target gene to evaluate the specificity and sensitivity of this assay. The RPA/CRISPR-Cas12a system has excellent specificity to distinguish D. aspalathi and D. caulivora from closely related species. The sensitivities of RPA/CRISPR-Cas12a-based fluorescence detection and lateral flow assay for D. aspalathi and D. caulivora are 14.5 copies and 24.6 copies, respectively. This assay can detect hyphae in inoculated soybean stems at 12 days after inoculation and has a recovery as high as 86% for hyphae-spiked soybean seed powder. The total time from DNA extraction to detection was not more than 60 min. The method developed for rapid detection of plant pathogens includes DNA extraction with magnetic beads or rapid DNA extraction, isothermal nucleic acid amplification at 39 °C, CRISPR-Cas12a cleavage reaction at 37 °C, and lateral flow assay or endpoint fluorescence visualization at room temperature. The RPA and CRISPR-Cas12a reagents can be preloaded in the microcentrifuge tube to simplify the procedures in the field. Both RPA and CRISPR-Cas12a reaction can be realized on a portable incubator, and the results are visualized using lateral flow strips or portable flashlight. This method requires minimal equipment and operator training, and has promising applications for rapid on-site disease screening, port inspection, or controlling fungal pathogen transmission in crop. © 2023 Society of Chemical Industry.

Remote detection of fungal pathogens in viticulture using laser-induced fluorescence: an experimental study on infected potted vines

IntroductionPathogenic fungi, such as Plasmopara viticola and Erysiphe necator, severely threaten the annual yield of grapes in both quantity and quality. In contrast to other crop production systems, fungicides are intensively applied in viticulture as a countermeasure. The goal of precision viticulture is to optimize vineyard performance as well as the environmental impact by reducing fungicides and applying different techniques and combined strategies. Therefore, new emerging technologies are required, including non-invasive detection, as well as monitoring and tools for the early and in-field detection of fungal development. MethodsIn this study, we investigated leaves of potted vines (Vitis vinifera cv. ‘Riesling’) and traced the development of the inoculated leaves using our new remote detection system vinoLAS®, which is based on laser-induced fluorescence spectroscopy. We ran a measurement campaign over a period of 17 days.ResultsWe were able to detect a leaf infection with P. viticola, the causal agent of downy mildew, between 5 and 7 days after inoculation. Our results provide evidence for a successful application of laser-based standoff detection in vineyard management in the future. Thus, the vinoLAS system can serve as a model technology for the detection of pathogenic disease symptoms and thus monitoring complete vineyard sites. This allows for early countermeasures with suitable crop protection approaches and selected hot-spot treatments.DiscussionAs P. viticola is considered one of the most damaging fungi in European viticulture, disease mapping via this monitoring tool will help to reduce fungicide applications, and will, therefore, support the implementation of the European Green Deal claims.

Detection of plant pathogenic fungi by a fluorescent sensor array

Detection and identification of plant pathogenic fungi have relevant implications for both food safety and human health, and require fast and easy-to handle diagnostic techniques. In this context, we report the design and development of a fluorescent array-based sensor, containing different organic receptors, able to detect selectively Penicillium italicum, Alternaria alternata and Fusarium sacchari. The detection of these pathogenic fungi can be performed using a smartphone, leading to the first example of detection of microscopic fungal plant pathogens by fluorescent array using a smartphone as detector. This study represents a proof-of-concept for the realization of practical sensoristic devices able to detect pathogens in plant products and food or in environmental samples, in order to guarantee food and human health security.

Brassicaceae Fungi and Chromista Diseases: Molecular Detection and Host–Plant Interaction

Brassicaceae plants cover a large number of species with great economic and nutritional importance around the world. The production of Brassica spp. is limited due to phytopathogenic fungal species causing enormous yield losses. In this scenario, precise and rapid detection and identification of plant-infecting fungi are essential to facilitate the effective management of diseases. DNA-based molecular methods have become popular methods for accurate plant disease diagnostics and have been used to detect Brassicaceae fungal pathogens. Polymerase chain reaction (PCR) assays including nested, multiplex, quantitative post, and isothermal amplification methods represent a powerful weapon for early detection of fungal pathogens and preventively counteract diseases on brassicas with the aim to drastically reduce the fungicides as inputs. It is noteworthy also that Brassicaceae plants can establish a wide variety of relationships with fungi, ranging from harmful interactions with pathogens to beneficial associations with endophytic fungi. Thus, understanding host and pathogen interaction in brassica crops prompts better disease management. The present review reports the main fungal diseases of Brassicaceae, molecular methods used for their detection, review studies on the interaction between fungi and brassicas plants, and the various mechanisms involved including the application of omics technologies.

Application of direct PCR for phylogenetic analysis of Fusarium fujikuroi species complex isolated from rice seeds.

Plant pathogenic fungi cause severe yield losses and mycotoxin contamination in crops. The precise and rapid detection of fungal pathogens is essential for effective disease management. Sequencing universal DNA barcodes has become the standard method for the diagnosis of fungal diseases, as well as for identification and phylogenetic analysis. A major bottleneck in obtaining DNA sequence data from many samples was the laborious and time-consuming process of sample preparation for genomic DNA. Here, we describe a direct PCR approach that bypasses the DNA extraction steps to streamline the molecular identification of fungal species. Using a direct PCR approach, we successfully sequenced the nuclear ribosomal internal transcribed spacer (ITS) region for the representatives of major fungal lineages. To demonstrate the usefulness of this approach, we performed a phylogenetic analysis of the Fusarium fujikuroi species complex, which causes bakanae ("foolish seedling") disease of rice and mycotoxin contamination. A total of 28 candidate strains were isolated from rice seeds in the Republic of Korea, and the identity of the isolates was determined using the DNA sequence of both ITS and translation elongation factor 1-α regions. In addition, 17F. fujikuroi isolates were examined for fumonisin (FB) production in rice medium using an enzyme-linked immunosorbent assay. Phylogenetic and toxigenic analyses showed that the F. fujikuroi strains could be distinguished into two groups: FB producers (B14-type) and non-producers (B20-type). These results will accelerate the molecular identification of fungal pathogens and facilitate the effective management of fungal diseases.

High-throughput and point-of-care detection of wheat fungal diseases: Potentialities of molecular and phenomics techniques toward in-field applicability

The wheat crop is one of the most cultivated and consumed commodities all over the world. Fungal diseases are of particular concern for wheat cultivation since they cause great losses and reduced quality, and also for the accumulation of toxin compounds into the final product. In this scenario, optimal disease management strategies are a key point to boosting food production and sustainability in agriculture. Innovative and point-of-care diagnostic technologies represent a powerful weapon for early detection of fungal pathogens and preventively counteract diseases on wheat with the aim to drastically reduce the fungicides as inputs. Indeed, in-field diagnostics devices are fast, sensitive, and ready-to-use technologies able to promptly detect a low inoculum concentration even at the pre-symptomatic stage of the disease. Promising isothermal molecular and phenomics-based methods have been developed to detect wheat fungal pathogens directly in the field. Such technologies could be potentially coupled to directly detect the presence of a certain pathogen and indirectly disclose the plant-pathogen interactions since spectral-based methodologies detect host perturbations following the infection. The present review reports the main in-field isothermal molecular-based and phenomics-based detection technologies for fungal pathogens in wheat discussing their advantages, disadvantages, and potential applications in the near future.

Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus.

IntroductionMultiplex quantitative polymerase chain reaction (qPCR) methods for the detection of Aspergillus spp. based only on SYBR Green and melting curve analysis of PCR products are difficult to develop because most targets are located within ITS regions. The aim of this study was to adapt our previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis to provide a qPCR method.MethodsA SYBR Green-based real-time PCR assay was optimized to detect A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus in a multiplex assay and applied to cultured fungi and spiked plasma.ResultsDifferent melting temperatures allowed identification of all five pathogens and discrimination between them, even in samples with low amounts of fungal gDNA (from 1.3 to 33.0 pg/μL), which has been reported previously as problematic. No false-positive results were obtained for non-target species, including bacteria and human DNA. This method allowed detection of fungal pathogens in human plasma spiked with fungal DNA and in coinfections of A. niger/R. arrhizus.DiscussionThis work provides evidence for the use of a qPCR multiplex method based on SYBR Green and melting curve analysis of PCR products for the detection of A. fumigatus, A. flavus, A. niger, A. terreus, and R. arrhizus. The proposed method is simpler and less expensive than available kits based on fluorescent probes and can be used for aiding diagnosis of the most relevant invasive filamentous fungi, particularly in low-income health care institutions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40291-022-00595-1.

Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications.

The increasing incidence and changing epidemiology of invasive fungal infections continue to present many challenges to their effective management. The repertoire of antifungal drugs available for treatment is still limited although there are new antifungals on the horizon. Successful treatment of invasive mycoses is dependent on a mix of pathogen-, host- and antifungal drug-related factors. Laboratories need to be adept at detection of fungal pathogens in clinical samples in order to effectively guide treatment by identifying isolates with acquired drug resistance. While there are international guidelines on how to conduct in vitro antifungal susceptibility testing, these are not performed as widely as for bacterial pathogens. Furthermore, fungi generally are recovered in cultures more slowly than bacteria, and often cannot be cultured in the laboratory. Therefore, non-culture-based methods, including molecular tests, to detect fungi in clinical specimens are increasingly important in patient management and are becoming more reliable as technology improves. Molecular methods can also be used for detection of target gene mutations or other mechanisms that predict antifungal drug resistance. This review addresses acquired antifungal drug resistance in the principal human fungal pathogens and describes known resistance mechanisms and what in-house and commercial tools are available for their detection. It is emphasized that this approach should be complementary to culture-based susceptibility testing, given the range of mutations, resistance mechanisms and target genes that may be present in clinical isolates, but may not be included in current molecular assays.

Host immune responses in the central nervous system during fungal infections.

Fungal infections in the central nervous system (CNS) cause high morbidity and mortality. The frequency of CNS mycosis has increased over the last two decades as more individuals go through immunocompromised conditions for various reasons. Nevertheless, options for clinical interventions for CNS mycoses are still limited. Thus, there is an urgent need to understand the host-pathogen interaction mechanisms in CNS mycoses for developing novel treatments. Although the CNS has been regarded as an immune-privileged site, recent studies demonstrate the critical involvement of immune responses elicited by CNS-resident and CNS-infiltrated cells during fungal infections. In this review, we discuss mechanisms of fungal invasion in the CNS, fungal pathogen detection by CNS-resident cells (microglia, astrocytes, oligodendrocytes, neurons), roles of CNS-infiltrated leukocytes, and host immune responses. We consider that understanding host immune responses in the CNS is crucial for endeavors to develop treatments for CNS mycosis.

Loop-Mediated Isothermal Amplification for Detection of Plant Pathogens in Wheat (Triticum aestivum).

Wheat plants can be infected by a variety of pathogen species, with some of them causing similar symptoms. For example, Zymoseptoria tritici and Parastagonospora nodorum often occur together and form the Septoria leaf blotch complex. Accurate detection of wheat pathogens is essential in applying the most appropriate disease management strategy. Loop-mediated isothermal amplification (LAMP) is a recent molecular technique that was rapidly adopted for detection of plant pathogens and can be implemented easily for detection in field conditions. The specificity, sensitivity, and facility to conduct the reaction at a constant temperature are the main advantages of LAMP over immunological and alternative nucleic acid-based methods. In plant pathogen detection studies, LAMP was able to differentiate related fungal species and non-target strains of virulent species with lower detection limits than those obtained with PCR. In this review, we explain the amplification process and elements of the LAMP reaction, and the variety of techniques for visualization of the amplified products, along with their advantages and disadvantages compared with alternative isothermal approaches. Then, a compilation of analyses that show the application of LAMP for detection of fungal pathogens and viruses in wheat is presented. We also describe the modifications included in real-time and multiplex LAMP that reduce common errors from post-amplification detection in traditional LAMP assays and allow discrimination of targets in multi-sample analyses. Finally, we discuss the utility of LAMP for detection of pathogens in wheat, its limitations, and current challenges of this technique. We provide prospects for application of real-time LAMP and multiplex LAMP in the field, using portable devices that measure fluorescence and turbidity, or facilitate colorimetric detection. New technologies for detection of plant pathogen are discussed that can be integrated with LAMP to obtain elevated analytical sensitivity of detection.

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Direct identification of fungi associated with indoor and outdoor ornamental plants by utilizing PCR assay

Review on Semiotics of Biosensors

Biosensors are the devices used to capture and keep a record of the biological signals. They later convert it into the electrical signals. They are composed with the combinations of various biological entities like; DNA, RNA, and Proteins. Amperometric, Surface Plasmon Resonance, DNA phage are the most commonly used sensors being employed today. Various reviews unveil the usage of eukaryotic microorganisms to plan the entire cell biosensors (WCB) to detect heavy metal pollutants. Few theories have also been introduced covering the screen printing theories with special emphasis on the technical impacts of SPE and its configurations. The biosensors dependent on engendering the surface. Plasmon resonances (SPRs) in films are the all-around perceived Plasmon biosensors with incredible potential for new advances like the film based SPR detecting. The most recent and impactful applications came forward were based on Bio-medical: Glucose Enzymes like Glucose oxidase and Glucose dehydrogenase, Bacterial Monitoring, Fungal Pathogen Detection, and Polychlorinated biphenyls. Biosensors have been promising over the most recent couple of years in the exact discovery of the natural mixtures in food and soil by the use of immunological biosensors. This monitoring of antigen-antibody interaction uses piezoelectric transducers.

Investigation and rapid detection of fungal pathogens causing mulberry sclerotial disease in south‐west China

AbstractMulberry sclerotial disease is a destructive disease that afflicts mulberry fruits throughout the world. In recent years, the impact of mulberry sclerotial disease has increased with the development of the mulberry fruit industry. Ciboria carunculoides, C. shiraiana, and Scleromitrula shiraiana are all pathogens causing mulberry sclerotial disease, but the dominant pathogen may be different in different mulberry planting areas. In this study, we investigated the causal agents of mulberry sclerotial disease in Chongqing and Sichuan, the main mulberry planting areas in south‐west China. The results showed that C. carunculoides was the dominant pathogen in the investigated areas, and the disease incidence rate on some widely cultivated mulberry varieties exceeded 90%. Based on the differences in internal transcriber spacer sequences of different pathogens, we developed a method using cleaved amplified polymorphic sequences (CAPS) from digestion with the three restriction enzymes HindIII, EcoRI, and MluI for rapid detection of pathogens on mulberry fruits. Use of this method confirmed that the diseased fruits of some mulberry varieties were infected by a mixture of C. carunculoides and S. shiraiana. Long‐fruit mulberry, which was considered to be resistant to sclerotial disease in the past, was also infected by C. carunculoides. Diseased fruit infected by C. shiraiana or by S. shiraiana alone were not detected. Our results suggest that C. carunculoides is probably the dominant pathogen causing mulberry sclerotial disease in south‐west China. The CAPS method for rapid detection of pathogens could assist diagnosis, epidemiology, and disease monitoring.

Detection of maize seed-borne fungi and induce resistance against both of Aspergillus niger and Fusarium verticillioides

The seeds of six cultivars of maize were screened by both blotter and agar methods for detection of fungal pathogens, and for testing the transmission of seed-borne fungi using test tube agar. Results showed that corn seeds infected by many pathogens included Fusarium, Aspergillus, Nigrospora, Macrophomina and Penicillium sp. To confirm the identification of isolates at the species level, using the internal transcribed spacer region (ITS) from rDNA as a barcode, our isolates were similar (99%) to the international isolates. Phylogenetic analysis of five isolates of F. verticillioides revealed correlations between national isolates compared with two international isolates sequenced in GenBank. Evaluation of salicylic acid as a risk-free antifungal chemical against both F. verticillioides and A. niger is also being done. In vitro studies have found salicylic acid to inhibit linear growth from 5 to10 mM. Based on in vivo studies, it helped increase root and shoot system dry weights from 5.6–37.4% and 9.1–59.5%, respectively.

Molecular detection of fungal pathogens and induction of phytoimmunity using bioinoculants

The present review deals with the serological and molecular techniques available for detection of fungal pathogens in plants, followed by management practices using bioinoculants which can induce resistance in the hosts. Specific detection of pathogens in the plant tissues even before the appearance of symptoms are done either through protein based immunodiagnostic techniques or DNA/RNA based molecular techniques. Immunotechniques depend on specific interaction between antigen of the pathogen or infected tissues and antibodies prepared against the specific antigen. Positive reactions are detected through either qualitative or quantitative techniques. Among the immunodiagnostic techniques, the most widely and commonly used is the enzyme linked immunosorbent assay (ELISA), Dot blot, Western blot and Immunofluorescence. The molecular techniques are predominantly PCR based and include different variations with increasing sensitivity such as nested PCR, multiplex PCR, reverse transcriptase PCR, real time PCR, loop-mediated isothermal amplification (LAMP) and nano based PCR. Thus, by use of one or more sophisticated techniques it is possible to detect pathogens rapidly and specifically. Pathogen detection is generally followed by management techniques, and in the current scenario ecologically safe bioinoculants are used for induction of immunity in the plants. There are several bioinoculants based on beneficial microorganisms such as plant growth promoting bacteria, biocontrol agents and arbuscular mycorrhizal fungi. Application of these microorganisms, singly or as a consortium, has been reported to induce immunity in plants. Induced systemic resistance (ISR) operates through signal molecules and pathways involving jasmonate and ethylene, and in very few cases, salicylate. Upregulation of these pathways leading to enhanced accumulation of antimicrobial compounds (phytoalexin) help in warding off the pathogen during subsequent attack.

Application of single-cell real-time imaging flow cytometry in rapid detection of pathogenic fungi in clinical liquid specimens

Rapid and direct observation of fungal spores or hyphae in clinical liquid specimens poses a challenge for the diagnosis of invasive fungal infection. To allow rapid detection of fungal pathogens, we designed a new method of fungal cell detection involving double fluorescence staining with calcium fluorescent white (CFW) and SYTOX green combined with single-cell real-time imaging flow cytometry (IFC). IFC allowed quick detection and analysis of detailed morphology of the spores and pseudohyphae of Candida albicans, and small hyphae and typical truncated large mycelia of Aspergillus fumigatus. Further, cell sorting based on fluorescence, the width-to-height ratio and bright-field parameters preferentially identified spores or hyphae with a typical cell wall. The specificity and overall coincidence rate of IFC for fungi detection in common clinical samples were 100% and 98.18%, respectively. Moreover, the detection rate by IFC (102/105, 97.14%) was significantly higher (P = 0.002) than that by wet mount method (89/105, 84.5%). Therefore, IFC is a reliable diagnostic method with a high potential for application for rapid diagnosis of fungal infection in the clinic.