Identification Of Fungal Pathogens Research Articles

Pulmonary Aspergillosis: An Observation from a Tertiary Care Hospital of Western Uttar Pradesh

Background: Aspergillus is the most frequently occurring fungal infection in immunocompromised patients with a high mortality rate. Invasive pulmonary aspergillosis is the most serious entity on the spectrum of pulmonary aspergillosis. Limited studies on the occurrence of pulmonary aspergillosis from this geographical area prompted us to carry out this study. Materials and Methods: This hospital-based cross-sectional observational study was carried out in the Postgraduate Department of Microbiology of a tertiary care medical teaching hospital in western Uttar Pradesh for one year. A total of 1032 respiratory samples were subjected for isolation and identification of fungal pathogens by direct microscopic examination (KOH mount) and culture on Sabouraud dextrose agar. Results: The isolation rate of pulmonary aspergilli was 6.97%. The majority of the respiratory samples received were sputum 900 (87.2%) followed by bronchoalveolar lavage fluid 132 (12.7%). The pulmonary pathology was predominantly seen in the age group of 41–50 years (25%). Male patients (69%) were predominant. The majority of the samples (50%) were from the respiratory medicine ward followed by the respiratory medicine ICU (25%). Aspergillus fumigatus (58%) was the predominant species isolated followed by Aspergillus flavus (25%). Conclusion: Isolation of Aspergillus species from patients of pulmonary pathology needs to be correlated clinically to rule out colonization as they may need proper antifungal treatment. Indiscriminate use of antifungals may lead to the emergence of antifungal drug resistance.

Nanopore sequencing in surveillance of fungicide resistance

By detecting plant pathogens as early as possible, swift action can be taken to mitigate the spread of disease. Monitoring fungicide is another key part of effective disease control as pathogens can develop resistance to fungicides, rendering them ineffective. The area of plant disease management is advancing apace thanks to innovative research incorporating technical advances in detection and monitoring. For example, Dr Semcheddine Cherrad, Research and Development Manager at Conidia Coniphy, is conducting innovative studies on microbiological assessment and contamination control. The research team at Conidia Coniphy has been working on the detection of plant pathogens and monitoring of fungicide resistance for over 15 years and the teamâ–™s collective efforts in bioassay and molecular analysis, combined with the adoption of new technologies and processes is overcoming challenges in the field. For example, the researchers are utilising new generation sequencing (NGS) with long-read sequencing to generate huge datasets on DNA molecules of genes coding for proteins targeted by fungicides. The team has developed a tool for mould identification called ID-Fungi Plates (IDFP). It is a user-friendly medium that accelerates the identification of fungal pathogens by Maldi-tof. They have also developed a software called FUNGI MESURE, which can quantify fungal spores in samples, providing an accurate overview of fungal measures and facilitating lab routine tasks of methods with fungi. In addition, a database established by the team called FUNGI DNA supports the identification and study of fungal strains through DNA analysis.

Identification of Pathogenic Fungi Causing Postharvest Disease of Banana Fruit (Musa acuminata Cavendish Subgroup) in Ho Chi Minh City, Vietnam

ABSTRACTBanana (Musa acuminata Cavendish Subgroup) cultivation plays a pivotal role in Vietnam's economy. However, the presence of postharvest diseases poses significant challenges as they adversely affect the quality and market value of the crop. The objective of this study was to identify the microbial strains responsible for these diseases through phylogenetic analysis and morphological characterisation. Four fungal isolates including Colletotrichum musae, C. gloeosporioides, Fusarium musae and Lasiodiplodia theobromae were identified as the primary causal agents of fruit rot in postharvest bananas. Re‐infection tests, which were conducted following Koch's postulates on banana samples, confirmed the isolates' significant pathogenicity, leading to weight loss, colour alteration, firmness reduction and acidity changes in the fungal‐infected bananas. These findings hold profound implications for enhancing our understanding of banana diseases in the postharvest stage, which can contribute to the development of effective control measures and mitigation strategies in the future.

Uncovering Fusarium Species Associated with Fusarium Wilt in Chickpeas (Cicer arietinum L.) and the Identification of Significant Marker–Trait Associations for Resistance in the International Center for Agricultural Research in the Dry Areas’ Chickpea Collection Using SSR Markers

Enhancing plants’ resistance against FW is crucial for ensuring a sustainable global chickpea production. The present study focuses on the identification of fungal pathogens and the assessment of ninety-six chickpea samples for Fusarium wilt from the International Center for Agricultural Research in the Dry Areas (ICARDA)’s collection. Eight fungal isolates were recovered from the symptomatic chickpeas. Polyphasic identification was conducted by comparing the internal transcribed spacer region (ITS), the elongation factor 1-α (tef1-α), and beta-tubulin (tub2). Among them, Neocosmospora solani, N. nelsonii, N. falciformis, N. brevis, Fusarium brachygibbosum, and F. gossypinum were identified. An analysis of the genetic diversity of chickpeas, using 69 polymorphic simple sequence repeat (SSR) markers, revealed a total of 191 alleles across all markers, with, on average, each SSR marker detecting approximately 2.8 alleles. A STRUCTURE analysis delineated lines into two distinct sub-groups (K = 2). Association mapping, using the generalized linear model (GLM) and mixed linear model (MLM) approaches, identified six and five marker–trait associations (MTAs) for FW resistance, respectively. Notably, these TA42, TA125 (A) and TA125 (B), TA37, and TAASH MTAs, commonly found in both models, emerge as potential candidates for the targeted enhancement of FW resistance in chickpeas. To our knowledge, this study represents an inaugural report on the association mapping of genomic loci governing FW resistance in chickpeas from the ICARDA’s accessions.

DETEKSI DAN IDENTIFIKASI PATOGEN CENDAWAN PADA UMBI KENTANG DI SULAWESI SELATAN

Potato (Solanum tuberosum L.) is an important horticultural commodity in Indonesia and the world, but its production has decreased due to attacks by Plant Pest Organisms including fungal pathogens. This study aims to detect and identify fungal pathogens found in potato tubers in potato-producing centers in South Sulawesi. Isolation of the fungus was carried out by the Blotter test method, namely incubating potato tubers on moist filter paper, then the fungal pathogens that appeared were identified. The results showed that the detection and identification of fungal pathogens found in potato tubers in South Sulawesi included the fungal Fusarium sp., Alternaria solani and Aspergillus sp. The percentage of fungal pathogens on potato tubers was dominated by the attack of the fungus Fusarium sp. with an average percentage of 57.5% (medium) in the Enrekang Potato sample, Gowa 62.5% (medium), 52.5% (medium) in the Bantaeng potato tuber sample. Then it was dominated by the fungus Aspergillus sp, on potato tubers Enrekang 47.5% (medium), Gowa 45% (medium) and Bantaeng 42.5% (medium) and the lowest attack rate was the attack of the fungus Alternaria solani (22.5 %) of potato samples from Bantaeng.

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.