Identification Of Fungal Pathogens Research Articles

Isolation and identification of fungal pathogens of post-harvest rot of tomato fruit in Biu Markets, Biu Local Government Area of Borno State

Isolation and identification of fungal pathogens of post-harvest rot of tomato fruit in Biu Markets, Biu Local Government Area of Borno State

Molecular Identification of Fungal Pathogens of Date Palm Root Rot in Egypt

Molecular Identification of Fungal Pathogens of Date Palm Root Rot in Egypt

Identification of fungal pathogens in otomycosis and their drug sensitivity in Monastir, Tunisia

Identification of fungal pathogens in otomycosis and their drug sensitivity in Monastir, Tunisia

Antagonistic Effect of Endophytic Fungi Isolated from Nerium olender against Rhizoctonia solani

Objective: To investigate the population of endophytic fungi accompanying to Nerium olender plant. Materials and Methods: The study was conducted on three different sites in two governorates for three consecutive months with five samples from each site. The identification of the fungi was carried out using microscopic and molecular methods while the isolation, identification of fungal pathogen and preparation of aqueous extract were also successfully done. Results: 14 species of fungi (Penicillium notatum, Rhizopus nigricans, Rhizopus stolonifera, Fusarium Solani, Fusarium oxysporum, Alternaria sp., Cladosporium sp .and Curvularia sp.), while the other sex species were identified by DNA investigation method and where (Aspergillus spp. (40%), Penicillium spp. (20%), Rhizopus spp. (12%), Fusarium spp. (10%), Alternaria sp. (4%), Mucor circinelloides (4%), Neurospora Crassa (3%), Cladosporium sp. (1%) and Curvularia sp. (1%). Conclusions: The highest percentage of inhibition was for fungi Aspergillus Flavus, Penicillium commune and Mucor circinelloides which amounted to 100% and from the third day of growth until the completion of the control dish, then Aspergillus niger and Rhizopus stolonifera, with a 90% on the seventh day of growth, and Penicillium commune, Rhizopus nigricans with a rate ranging between 82-68%, while the rest of the fungi showed an antagonistic effect, but with rates less than 50%.

Molecular Identification of Fungal Pathogens causing Feather Infection in Parrots Suffering from the Psittacine Beak and Feather Disease (PBFD) Virus

Molecular Identification of Fungal Pathogens causing Feather Infection in Parrots Suffering from the Psittacine Beak and Feather Disease (PBFD) Virus

Isolation, phenotypic characterization and molecular identification of fungal pathogens of sweet pepper (Capsicum annuum L.) Fruit in Sokoto State Nigeria

Isolation, phenotypic characterization and molecular identification of fungal pathogens of sweet pepper (Capsicum annuum L.) Fruit in Sokoto State Nigeria

Identification of fungal pathogens causing fruit tree dieback in British Columbia

Field surveys were conducted in British Columbia fruit tree growing regions to determine the incidence of dieback and to identify the main fungal species causing cankers. Fruit trees showing cankers and dieback symptoms were recorded from 94% of orchards and 5.5% of trees surveyed. Overall, higher dieback incidence was observed in cherry than apple with 33% of cherry blocks showing between 5% and 26% of trees affected. Morphological studies along with DNA sequencing of the internal transcribed spacer (ITS) including the 5.8S rDNA, and parts of the translation elongation factor 1-α (TEF1), beta-tubulin (TUB2), and actin (ACT1) genes, identified seven fungi for the first time in fruit trees in Canada, including Calosphaeria pulchella, Cytospora parasitica, Cytospora populicola, Cytospora sorbicola, Ilyonectria robusta, Nectria dematiosa, and Phaeoacremonium minimum. In addition, this study reports for the first time Diplodia mutila and Diplodia seriata from cankers in sweet cherry in Canada. The already known fungal pathogens Neofabraea perennans and Neonectria ditissima were also identified. Pathogenicity studies showed N. ditissima and C. sorbicola to cause the largest vascular lesions in apple and cherry, respectively. This study identified the main fungal pathogens causing tree fruit cankers and dieback in British Columbia providing important information for the development of effective control strategies.

Residual neural networks in single instance-driven identification of fungal pathogens

The rise in fungal infections, attributed to various factors including medical interventions and compromised immune systems, necessitates rapid and accurate identification methods. While traditional mycological diagnostics are time-consuming, machine learning offers a promising alternative. Nevertheless, the scarcity of well-curated datasets is a significant obstacle. To address this, a novel approach for identifying fungi in microscopic images using Residual Neural Networks and a subimage retrieval mechanism is proposed, with the final step involving the implementation of majority voting. The new method, applied to the Digital Images of Fungus Species database, surpassed the original patch-based classification using Convolutional Neural Networks, obtaining an overall classification accuracy of 94.7% compared to 82.4% with AlexNet FV SVM. The observed MCC metric exceeds 0.9, while AUC is near to one. This improvement is attributed to the optimization of hyperparameters and top layer architecture, as well as the effectiveness of the Mish activation function in ResNet-based architectures. Noteworthy, the proposed method achieved 100% accurate classification for images from 8 out of 9 classes after majority voting and is high resistant to overfitting, highlighting its potential for rapid and accurate fungal species identification in medical diagnostics and research.

987. Identification of Fungal Pathogens In Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by Targeted Next-Generation Sequencing (NGS) Technology

Abstract Background Histological presence of fungal organisms associated with tissue damage is the hallmark for invasive fungal diseases. Rapid and accurate identification (ID) of these fungal pathogens present is critical for initiating appropriate treatment without delay. We investigated an automated sample-to-answer targeted NGS workflow for the ID of fungi directly from FFPE tissues. Methods 170 FFPE tissues (93 cases) from 2002 to 2022 at Johns Hopkins Hospital were selected based on histologic findings of the presence of fungal pathogens. Samples were run by PathoKey SQ Flex assay, (Vela Diagnostics) which includes automated extraction, library preparation and emulsion PCR followed by NGS on a Sentosa SQ301 and analyzed using Vela’s curated database. Two negative controls (NCs) were included on each run. To compare NGS results, 51 samples were extracted using the MagMAX™ FFPE DNA/RNA Ultra Kit (Applied Biosystems) followed by pan-fungal PCR (ITS1/ITS2/D1D2) and Sanger sequencing (SS) and analyzed through NCBI nucleotide blast for ID. Results 116/170 (68%) samples had culture results: 69/116 (59%) were concordant with Vela NGS (VS) ID to the genus/species level; 18/28 (64%) samples were concordant with SS ID to the genus/species level. Among 54 samples with no/negative culture results: 26/54 (48%) were presumptive Mucorales, of which 23/26 (88.5%) were supported by VS ID and 5/12 (42%) supported by SS ID; 12/54 (22%) were presumptive aspergillus-like hyphae fungi, of which 10/12 (83%) were supported by VS ID and 4/9 (44%) supported by SS ID. 15/170 (8.8%) samples had no VS ID due to QC failures, and 15/26 (58%) with no VS ID had no organism seen or scant amount of fungal load in the histology slide. Conclusion VS detected fungal organisms in 76% (129/170) of FFPE tissues, of which it correctly ID-ed pathogens down to the genus/species level in 94% (102/129) of samples. VS is more sensitive to pathogen ID than SS. Samples with discordant or no results by VS were mostly due to small tissue input or low fungal loads present in histology. Environmental or human skin flora contamination reads overshadowing possible pathogens were observed in few cases; therefore, it is critical to include NCs in each run to distinguish them from clinically significant fungal pathogens. Disclosures Marissa C. Totten, MA, Vela Diagnostics: Grant/Research Support Pramila Ariyaratne, n/a, Vela Diagnostics: Employee Tony Zhang, n/a, Vela Diagnostics: Employee Sean Zhang, MD, PhD, Applied BioCode: Grant/Research Support|IMMY Diagnostics: Grant/Research Support|KARIUS: Advisor/Consultant|Pearl Diagnostics: Grant/Research Support|Scanogen: Grant/Research Support|T2 biosystems: Advisor/Consultant|Vela Diagnostics: Grant/Research Support Charlie Lee, Ph.D, Vela Diagnostics: Employee

Rapid and Accurate Diagnosis of Dermatophyte Infections Using the DendrisCHIP® Technology.

Dermatophytosis is a superficial fungal infection with an ever-increasing number of patients. Culture-based mycology remains the most commonly used diagnosis, but it takes around four weeks to identify the causative agent. Therefore, routine clinical laboratories need rapid, high throughput, and accurate species-specific analytical methods for diagnosis and therapeutic management. Based on these requirements, we investigated the feasibility of DendrisCHIP® technology as an innovative molecular diagnostic method for the identification of a subset of 13 pathogens potentially responsible for dermatophytosis infections in clinical samples. This technology is based on DNA microarray, which potentially enables the detection and discrimination of several germs in a single sample. A major originality of DendrisCHIP® technology is the use of a decision algorithm for probability presence or absence of pathogens based on machine learning methods. In this study, the diagnosis of dermatophyte infection was carried out on more than 284 isolates by conventional microbial culture and DendrisCHIP®DP, which correspond to the DendrisCHIP® carrying oligoprobes of the targeted pathogens implicated in dermatophytosis. While convergence ranging from 75 to 86% depending on the sampling procedure was obtained with both methods, the DendrisCHIP®DP proved to identify more isolates with pathogens that escaped the culture method. These results were confirmed at 86% by a third method, which was either a specific RT-PCR or genome sequencing. In addition, diagnostic results with DendrisCHIP®DP can be obtained within a day. This faster and more accurate identification of fungal pathogens with DendrisCHIP®DP enables the clinician to quickly and successfully implement appropriate antifungal treatment to prevent the spread and elimination of dermatophyte infection. Taken together, these results demonstrate that this technology is a very promising method for routine diagnosis of dermatophytosis.

Identification of common fungal pathogens of dragon fruit (Hylocereus undatus) in Sarangani Province, Philippines

Fungal disease is one of the major challenges that affects yield quality and profit in dragon fruit production today, as it causes a considerable amount of production loss. In this study, fungal pathogens of dragon fruit (Hylocereus undatus) plants in Southern Philippines were identified. Sample collection was conducted in selected farms in Saranggani Province, Philippines. Diseased parts of the dragon fruit plants were collected and brought to the laboratory to culture its associated fungi. Grown fungi were pure cultured and subjected to pathogenicity testing using the detached stem method. A total of eight distinct fungal colonies were isolated from the collected dragon fruit samples. The pathogenicity test revealed that three of the isolates caused lesions in a healthy dragon fruit plant tissue, which turned yellow soft and watery as the infection progressed. The three pathogenic isolates were molecularly identified as Fusarium oxysporum (FRI3), Aspergillus minisclerotigenes (FRI2), and Fusarium incarnatum (FLI3). Interestingly, F. incarnatum and A. minisclerotigenes were new recorded pathogens of dragon fruit. Identification of fungal pathogens on economically important crops is an essential step in the development of strategies to address problems related to fungal diseases which, in turn, will help farmers to enhance their production.

Genetic variation of sequencing 18srDNA gene of Trichophyton mentagrophytes isolates

Background. Trichophyton species are considered the most frequent causative and considerable agents in infection concerns. Objective. The existing training designed to conduct the sequencing analysis of nucleotides of ITS1-5.8S-ITS2 for Trichophyton mentagrophytes. Methods. The isolation and identification of pathogenic fungi, Trichophyton mentagrophytes from clinical specimens was done based on the standard morphological and molecular methods. The genomic DNA of fungal isolates were extracted and purified to amplify with primers of 18S rRNA gene for detection and sequencing the nitrogenous bases to define the genetic variation among clinical isolates of Trichophyton mentagrophytes in compared with strains recorded in NCBI GenBank. Results. Eight isolates of Trichophyton mentagrophytes were isolated and identified from clinical specimen of dermatophytosis. The DNA sequencing analysis showed the presence of some genetic variation in nitrogenous bases between the local isolates and that recorded in locus of world NCBI GenBank. Multiple Alignment sequence using NCBI BLAST revealed the recoding a new mutant isolate among the local isolates based on DNA homology percent. Conclusion. The present results proved the successful the use of molecular methods in diagnosis the fungal isolates of Trichophyton mentagrophytes especially the sequencing analysis to find the genetically bases of these isolates.

Diversity of insect carried-fungi in chili (Capsicum annuum) crop at Banyumas District, Central Java Province, Indonesia

Abstract. Suroto A, Mugiastuti E, Tarjoko, Oktaviani E, Bahrudin M. 2023. Diversity of insect carried-fungi in chili (Capsicum annuum) crop at Banyumas District, Central Java Province, Indonesia. Biodiversitas 24: 3394-3406. This study aimed to determine the species of fungal pathogens that have the potency to be carried by various insects in endemic areas of chili (Capsicum annuum L.) disease and locations with high populations of chili insect vectors in the Banyumas District. The insect samples were collected from three chili planting locations in Banyumas District (Karanglewas, Sumbang, and Sokaraja Sub-districts) from March to November 2022. The following methods carried out the research: (i) determining the sampling location, (ii) insect sampling and identification, (iii) isolation and purification of fungal pathogen carried by insects, (iv) pathogenicity test, (v) identification of fungal pathogens based on morphology characters. The exploration of 13 villages in 3 (three) sub-districts of Banyumas District found 60 insects collected from healthy and diseased chili plants. Totally, six genera of fungi were isolated and identified, namely Fusarium, Pythium, Curvularia, Penicillium, Geotrichum, and Phytophthora. This is preliminary research on the interaction between plants, microbes, and insects. Understanding these aspects is essential, not only from an ecological perspective but also for improving the genetic quality of crops as well as for integrated pest management.

First record of Efibula tuberculata (Basidiomycota: Polyporales) isolated from sugarcane in Egypt

A new pathogenic fungus was noticed in 2020 for the first time as white rot on cuttings of sugarcane-grown fields of El-Mattana district (Luxor governorate), Kom-Ombo district (Asswan governorate) and Qus district (Qena governorate), Egypt. The causal organism was isolated from affected cuttings using Koch’s postulates, during 2021 and repeated at 2022, the relationship between the recovered isolate and the observed symptoms had been established. Artificially inoculated cuttings of sugarcane cultivar G.T. 54-9 gave symptoms similar to those appeared on naturally field-infected sugarcane cuttings. Morphological characters and molecular analysis, based on fungal internal transcribed spacer (ITS), were carried out to detect the recovered isolate. Identification of fungal pathogen revealed that Efibula tuberculata, Basidiomycota, was the causal organism of the white-rot of cuttings and roots of sugarcane. According to the available literature, this was the first report of occurring E. tuberculata isolate lux in Egypt and also as a sugarcane pathogen globally. Further studies to find the potent strategy to control, determine the susceptibility of different sugarcane cultivars and the fungus host range are needed.

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.

Early warning of Diaporthe infection in kiwifruit soft rot by plasmonic dimer-enhanced Raman spectroscopy.

Diaporthe genus is the dominant pathogens of kiwifruit soft rot with long incubation period and rapid onset and very hard to detect in advance. It is of great significance to develop point-of-care tests for disease prevention and field management. Here we screen a pair of genus-level primers for constructing plasmonic dimer Rayleigh/Raman spectroscopy, which enables rapid, specific, and sensitive detection of Diaporthe genus in real kiwifruits. Dark-filed Rayleigh scattering clearly visualizes the target-induced dimer assembly of nanoprobes. Plasmonic dimer-enhanced Raman spectroscopy has high specificity for 12 common isolates of Diaporthe genus in one batch samples with highly accurate results and much higher sensitivity than polymerase chain reaction (PCR). It realizes the recognition of Diaporthe infection at an early stage of 24h when the kiwifruits do not have any noticeable symptoms. It demonstrates a bright prospect of point-of-care tests for early warning of kiwifruit soft rot infection and quality control.

P180 Is gene sequencing only way forward?

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM IntroductionFungal peritonitis (FP) associated with peritoneal dialysis (PD) is an uncommon complication. Candida peritonitis accounts for 70%-90%. A total of 55 cases of Aspergillus peritonitis have been reported from 1968 to 2019 with A. fumigatus and A. niger as the most common isolates.FP is associated with high mortality and often results in discontinuation of PD and switching to hemodialysis (HD).ObjectivesTo identify and report the pathogen from peritoneal abscess.Methods and ResultsA 54-year-old post-menopausal lady, a known diabetic, hypertensive with chronic kidney disease (CKD) on PD for 2 years presents with acute onset breathlessness. She was dialyzed and improved. During her hospital stay, she developed a fever which was managed with intravenous antibiotics, piperacillin-tazobactam, teicoplanin, and fluconazole. The PD was bacterial culture negative. Patient improved on HD and was discharged.A total of 10 days later she was readmitted due to complaints of fever. Her PD fluid analysis was clear with high leucocyte count. The same was sent for the presence of acid-fast bacilli and aerobic bacterial culture, both of which were negative. Her blood was sent for automatic aerobic bacterial culture which yielded no growth. Patient developed pain in abdomen and PD catheter was removed. Abdominal Computed Tomography (CT) imaging revealed a thick-walled intra-peritoneal collection in the left subdiaphragmatic region. In view of persistent fever, antibiotics were changed to meropenem, metronidazole, and amikacin. Pus aspirated was received for aerobic bacterial culture and fungal culture. KOH—Calcofluor examination revealed septate fungal hyphae. Bacterial culture yielded Enterococcus species sensitive to penicillin and gentamicin. Patient was treated with amphotericin B and HD. Repeat aspirate also revealed septate fungal hyphae. However, on both occasions fungal cultures were negative. She improved symptomatically and was discharged.Sample was sent to Post Graduate Institute of Medical Education and Research, Chandigarh for the identification of the fungal pathogen directly from the sample using Gene sequencing. The pathogen was identified as A. niger.Conclusion Aspergillus peritonitis in a PD patient though rare is associated with high mortality and morbidity. They are often associated with concurrent or prior bacterial peritonitis. Treatment with antibiotics often predisposes to fungal peritonitis. Clinical presentation is similar to bacterial peritonitis, a high index of suspicion is warranted to establish the diagnosis. Identification of fungal pathogen to rule out species inherently resistant to certain classes of antifungals is critical for successful management. This is possible only by molecular methods, especially in culture-negative cases. In this case of culture-negative invasive fungal infection, the preliminary diagnosis was based on microscopic evidence of two consecutive samples. Gene sequencing helped us to confirm the diagnosis.

Identification of Pathogenic Fungi Causing Leaf Spot of Urtica cannabina and Malus sieversii in the Wild Fruit Forest of Tianshan Mountain, Xinjiang, China

Degradation of the wild apple trees in the wild fruit forest of Tianshan mountain of Xinjiang Province, China, has attracted great attention in recent years, and pathogens are believed to be an important responsible factor. We observed that Malus sieversii and its understory plant, Urtica cannabina, exhibited similar symptoms of leaf spot disease, and we suspect that they are caused by the same pathogens. DNA sequencing using ITS1 and ITS4 primers was applied to identify the pathogenic fungi from diseased leaves of U. cannabina and M. sieversii, which led to the identification of Alternaria sp. and Fusarium sp. as active pathogens causing same symptoms on leaves of both species. Our results implied that these two plants shared the same pathogenic fungi that cause leaf spot disease, and infection of the understory species U. cannabina might provide a reservoir of the pathogens which can attack M. sieversii and contribute at least in part, to the degradation of M. sieversii.

Surgical site infections after simultaneous pancreas kidney and pancreas transplantation in the Swiss Transplant Cohort Study

Among hospital-acquired infections, surgical site infections (SSIs) are frequent. SSI in the early post-transplant course poses a relevant threat to transplant recipients. To determine incidence, risk factors for SSI and its association with post-transplant outcomes and pancreas transplant (P-Tx) recipients. Adult simultaneous kidney-pancreas transplantation (SPK-T) and P-Tx recipients with a follow-up of at least 90 days were identified in the Swiss Transplant Cohort Study (STCS) dataset. Except for the categorization of SSIs according to Centers for Disease Control and Prevention (CDC) criteria, all other data were prospectively collected. Risk factors for SSI were investigated with logistic regression. A Weibull accelerated failure-time model was applied to address the impact of SSI on length of stay, correcting for transplant-related complications and delayed graft function. Of 130 transplant recipients, 108 SPK-Tx and 22 P-Tx, 18 (14%) individuals developed SSI within the first 90 days after transplantation. Deep incisional (seven, 38.9%) and organ/space infections (eight, 44.4%) predominated. In the majority of SSIs (11, 61.1%; two SSIs with simultaneous identification of fungal pathogens) bacteria were detected with Enterococcus spp. being most frequent. The median duration of hospitalization after transplantation was significantly longer in recipients with SSI (median: 26 days; interquartile range (IQR): 19-44) than in patients without SSI (median: 17 days; IQR: 12-25; P=0.002). In multivariate analysis, SSI was significantly associated with increased length of stay and prolonged the duration of hospitalization by 36% (95% confidence interval: 4-79). SSI after SPK-Tx and P-Tx occurred at a frequency of 14%. Among pathogens, Enterococcus spp. predominated. SSI was independently associated with a longer hospitalization after transplantation.

Identification of Pathogenic Fungi in Renal Transplant Patients by Conventional and Molecular Methods

Identification of Pathogenic Fungi in Renal Transplant Patients by Conventional and Molecular Methods