Universal Fungal Primers Research Articles

The ancestral environment of teosinte populations shapes their root microbiome

SummaryBackgroundThe composition of the root microbiome affects the host’s growth, with variation in the host genome associated with microbiome variation. However, it is not known whether this intra-specific variation of root microbiomes is a consequence of plants performing targeted manipulations of them to adapt to their local environment or varying passively with other traits. To explore the relationship between the genome, environment and microbiome, we sampled seeds from teosinte populations across its native range in Mexico. We then grew teosinte accessions alongside two modern maize lines in a common garden experiment. Metabarcoding was performed using universal bacterial and fungal primers to profile their root microbiomes.ResultsThe root microbiome varied between the two modern maize lines and the teosinte accessions. We further found that variation of the teosinte genome, the ancestral environment (temperature/elevation) and root microbiome were all correlated. Multiple microbial groups significantly varied in relative abundance with temperature/elevation, with an increased abundance of bacteria associated with cold tolerance found in teosinte accessions taken from high elevations.ConclusionsOur results suggest that variation in the root microbiome is pre-conditioned by the genome for the local environment (i.e. non-random). Ultimately, these claims would be strengthened by confirming that these differences in the root microbiome impact host phenotype, for example, by confirming that the root microbiomes of high-elevation teosinte populations enhance cold tolerance.

Peritoneal dialysis-associated peritonitis from pauci-septated mold: Life-threatening but curable

Two cases of PD-associated peritonitis due to Cunninghamella (C. bertholletiae and C. guizhouensis) were reported here with favorable outcomes, albeit presenting with septicemia. Both patients presented with classic features of bacterial peritonitis, cloudy effluent with a neutrophil predominance, followed by fever and septicemia/septic shock. The pathogen species were confirmed and verified by molecular phylogeny using universal and specific fungal primers. All isolations were susceptible/intermediately susceptible to amphotericin B but resistant to other antifungal agents, including triazoles, caspofungin, and terbinafine. Both cases were successfully treated with timely PD catheter removal and antifungal medications for 2–4 weeks.

Türkiye’nin kuzeybatısındaki bir plantasyonda yerli ve egzotik çam türlerinin ibrelerinde görülen fungal endofitler

The endophytic fungi present in needles of Pinus sylvestris, Pinus pinaster, Pinus nigra, Pinus taeda and Pinus radiata were investigated in Kerpe Research Forest, İzmit in 2016. Ten trees of each pine species were sampled systematically. Previous years green needles were sampled from the lower part of the canopy, from two equally spaced positions around the tree. Each needle was surface sterilized and cut into 0.5 cm sections then individually placed onto malt extract agar plates. DNA was ex-tracted from reprensentative seven isolates and amplified using primers ITS1 and ITS4 targeting the nuclear 5.8S rDNA gene and the two ITS regions flanked between 18S and 28S rDNA genes. Amplicons sequenced in both directions using the univer-sal fungal primers ITS1 and ITS4. Isolations from a total of 1000 needles (200 from each pine species) yielded 750 fungal isolates. Pestalotiopsis funerea (Desm.) Steyaert was isolated at the highest frequency followed by Acremonium sp., Cladio-sporum sp. and Cyclaneusma minus.

First report of a Panax notoginseng leaf disease caused by Alternaria alternata in China.

Panax notoginseng (Burk.) F. H. Chen is a perennial plant species in the family Araliaceae, and its roots and rhizome are precious materials for the production of traditional Chinese medicine. From April to June, 2018, new disease symptoms were detected on the mature leaves of 2- and 3-year-old Panax notoginseng (P. notoginseng) in Wenshan Autonomous Prefecture, Yunnan Province, China, and the disease incidence was about 10%-15% among the analyzed fields (3.6 ha, 23°49'46.99″ N, 104°06'12.99″ E, 1,631 m elevation). The diseased leaves had dark brown necrotic lesions (0.9-2.5 × 1.0-3.5 cm) and curled downward. As the disease progressed, the necrosis gradually spread along the vein to other leaf parts, eventually covering the whole leaf. In the late disease stage, the whole leaf was decayed and yellowed. For pathogen isolation, infected leaves (n=20) were surface sterilized in 1% sodium hypochlorite and washed with sterilized distilled water for 3 mins before being cut into smaller pieces (~1cm2), then placed onto potato dextrose agar (PDA) medium and incubated at 28°C under aseptic conditions for 3 days. The hypha around leaf discs were transferred onto the new PDA. A total of 20 colonies (SQ1~20) were obtained and purified by single spore culture for morphological characterization and molecular biological identification. The colonies of all isolates were nearly round, grayish white at the initial stage, and then turned to grayish brown. In addition, microscopic examination (100× magnification) of 20 isolates revealed dark, septate, and sparsely branched conidiophores as well as dark brown conidia with short conical beaks at their tip. Additionally, conidia (solitary or in short chains) were typically oval or club-shaped and had 0-2 longitudinal septa and 2-4 transverse septa (20-35 × 8-12 μm) (n = 50). Moreover, the conidia had a smooth or verrucose surface. Their morphological characteristics were similar to those descriptions given for members of section Alternaria by Lawrence et al. (2016). In order to further identify pathogenic species, genomic DNA was extracted from the colonies (SQ1~20) using a modified cetyltrimethylammonium bromide (CTAB) method (Loganathan et al. 2014). The sequences of internal transcribed spacer regions of ribosomal DNA (rDNA ITS) and partial RNA polymerase II second subunit gene (RPB2) were amplified by PCR using fungal universal primers ITS1/ITS4 (White et al. 1990) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. The DNA sequencing shows that ITS sequences from 20 isolates were totally same, and so did the RPB2 sequences (supplementary material). BLASTN analysis of NCBI database indicated that the RPB2 and ITS sequences have the highest nucleotide homology to A. Alternata ITS (MW008974) and RPB2 (LC132700), respectively. These two gene sequences were submitted to GenBank [Accession numbers ON075466 (ITS) and OP572232 (RPB2)]. Phylogenetic trees based on the combined ITS and RPB2 sequences were constructed by maximum parsimony method. The referenced ITS and RPB2 sequences of Alternaria were from three published articles (Rama et al. 2020; Sun et al. 2021; Wee et al. 2006). Phylogenetic analysis revealed that this isolate was clustered with A. alternata. Therefore, the morphology-based preliminary identification was verified by the phylogenetic analysis, and the isolate from diseased P. notoginseng leaves was A. alternata. To confirm its pathogenicity, the fungal isolate was assessed with 40 1-year-old healthy P. notoginseng plants in a greenhouse. Among them, the leaves of 20 of P. notoginseng plants were wounded using a sterile needle (seven or eight wounds per leaf) and then inoculated with 1mL conidial suspension (1 × 106 conidia/mL) prepared from 7-day-old fungal cultures grown on PDA medium. The inoculated plants were covered with plastic bags at 25°C for 24 h to maintain humidity, and then transferred to the greenhouse maintained at 28°C with a 16-h day/8-h night cycle and continuous misting. The other 20 control plants were only wounded and sprayed with sterile water. Typical necrotic lesions were detected on all of the inoculated P. notoginseng leaves cultivated in the greenhouse for 1 week post-inoculation. As the disease continued to develop, the necrotic lesions enlarged, and the infected leaves turned yellow and withered. These symptoms were similar to those observed on the naturally infected P. notoginseng. In contrast, the mock-inoculated control plants remained healthy. Furthermore, the fungus re-isolated from the infected P. notoginseng leaves in the pot experiment had similar morphological characteristics as the original strain. In addition, its genomic DNA was extracted for PCR analysis of ITS and RPB2 sequences, and the following DNA sequencing shows that the two DNA sequences were same as those of isolates SQ1~20, which confirmed that the re-isolated fungus was A. alternata. To the best of our knowledge, this is the first report of A. alternata causing a P. notoginseng leaf disease in China.

Comparison of in vitro activities of newer triazoles and classic antifungal agents against dermatophyte species isolated from Iranian University Hospitals: a multi-central study

BackgroundDermatophytes have the ability to invade the keratin layer of humans and cause infections. The aims of this study were the accurate identification of dermatophytes by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method and sequencing and comparison between the in vitro activities of newer and established antifungal agents against them.MethodsClinical specimens of patients from five Iranian university laboratories were entered in this study. Samples were cultured on sabouraud dextrose agar medium. For molecular identification, extracted DNAs were amplified by the universal fungal primers ITS1 and ITS4, and digested with MvaI enzymes. The antifungal susceptibility test for each isolate to terbinafine, griseofulvin, caspofungin, fluconazole, itraconazole, luliconazole, and isavuconazole was performed, according to the microdilution CLSI M38-A2 and CLSI M61 standard methods.ResultsTwo hundred and seven fungi species similar to dermatophytes were isolated of which 198 (95.6%) were dermatophytes by molecular assay. The most commonly isolated were Trichophyton mentagrophytes (76/198), followed by Trichophyton interdigitale (57/198), Trichophyton rubrum (34/198), Trichophyton tonsurans (12/198), Microsporum canis (10/198), Trichophyton simii (3/198), Epidermophyton floccosum (3/198), Trichophyton violaceum (2/198), and Trichophyton benhamiae (1/198). The GM MIC and MIC90 values for all the isolates were as follows: terbinafine (0.091 and 1 μg/ml), griseofulvin (1.01 and 4 μg/ml), caspofungin (0.06 and 4 μg/ml), fluconazole (16.52 and 32 μg/ml), itraconazole (0.861 and 8 μg/ml), isavuconazole (0.074 and 2 μg/ml), and luliconazole (0.018 and 0.25 μg/ml).ConclusionTrichophyton mentagrophytes, Trichophyton interdigitale, and Trichophyton rubrum were the most common fungal species isolated from the patients. luliconazole, terbinafine, and isavuconazole in vitro were revealed to be the most effective antifungal agents against all dermatophyte isolates.

Morphological and molecular diversity of Ganoderma spp. causal agent of basal stem rot of coconut in Southern dry tracts of Karnataka

Morphological and molecular diversity of Ganoderma species causing basal stem rot of coconut in Southern dry tracts of Karnataka, India was carried out during 2016-17. A total of 20 isolates were isolated from Chitradurga, Chikamagalore, Hassan and Tumkur districts of Karnataka and were identified based on morphological and molecular characteristics. Sporocarps and diseased root bits were found as good source for isolation of Ganoderma. In all the isolates there were high variability in cultural, morphological and molecular characteristics. The dendrogram generated from the cultural and morphological characteristics showed clear variations among Ganoderma isolates and formed two main clusters, one cluster consisted of 13 isolates and another cluster consisted of 7 isolates. Several isolates showed 100 per cent similarity in the morphological characters regardless of their geographical origin. All the Ganoderma isolates amplified a fragment of 650 bp with fungal universal primers (ITS1 and ITS4). The ITS gene sequences of five isolates viz., CG1 (MK 681870), CG7 (MK681871), CG11 (MK681872), CG14 (MK681873) and CG20 (MK681874) were deposited in NCBI gene bank. Taxonomic comparison of the isolates with NCBI database proved that the isolates were genetically related to Ganoderma spp. with 80-100 per cent identity. However, all the tested isolates could not amplify G. lucidum species specific markers which indicate its absence in the region. The phylogenetic analysis of the Ganoderma isolates (ITS1 and ITS4) of coconut with other known species of Ganoderma from GenBank emphasized the close relationship with India, China and Sri Lanka isolates. The isolate CG1 grouped with Ganoderma carnosum (KR 733545.1) with 98.97 per cent identity which is isolated from Sri Lanka and CG14 and CG20 grouped with G. applanatum (MF 072395.1) and G. gibbosum (OM 350473.1) with 98 to 99 per cent identity and CG7 and CG11 isolates of coconut grouped into distinct sub cluster and clearly indicated the species diversity in Ganoderma infecting coconut in Southern Karnataka.

The Occurrence of Stereum hirsutum Associated with Wood Rotting of Olive Trees in Iran

Abstract In summer 2021 (June–July), disease symptoms on trunks and shoots were observed in olive groves in the Tarom Sofla region, Qazvin province, Iran. The symptoms were light-pink discoloration and surface depression in the external layers of the wood. As the disease progressed, brown streaks of tissue appeared on the longitudinal sections of the wood. The identification of fungus was made based on PCR amplification of the rDNA-ITS region with the universal fungal primers ITS5 and ITS4. BLAST searches revealed 99.52% identity to Stereum hirsutum. Several species of basidiomycetes are known to live on wood as saprobionts or parasites. On olive trees, they cause white rot symptoms. Although they are not directly responsible for tree mortality; however, they can lead to structural deterioration of woody tissues. To the best of our knowledge, this is the first report of S. hirsutum associated with wood rotting of olive trees in the world.

Towards Improved Detection and Identification of Rust Fungal Pathogens in Environmental Samples Using a Metabarcoding Approach

The dispersion of fungal inocula such as the airborne spores of rust fungi (Pucciniales) can be monitored through metabarcoding of the internal transcribed spacer 2 (ITS2) of the rRNA gene in environmental DNAs. This method is largely dependent on a high-quality reference database (refDB) and primers with proper taxonomic coverage and specificity. For this study, a curated ITS2 reference database (named CR-ITS2-refDB) comprising representatives of the major cereal rust fungi and phylogenetically related species was compiled. Interspecific and intraspecific variation analyses suggested that the ITS2 region had reasonable discriminating power for the majority of the Puccinia species or species complexes in the database. In silico evaluation of nine forward and seven reverse ITS2 primers, including three newly designed, revealed marked variation in DNA amplification efficiency for the rusts. We validated the theoretical assessment of rust-enhanced (Rust2inv/ITS4var_H) and universal fungal (ITS9F/ITS4) ITS2 primer pairs by profiling the airborne rust fungal communities from environmental samples via a metabarcoding approach. Species- or subspecies-level identification of the rusts was improved by use of CR-ITS2-refDB and the Automated Oligonucleotide Design Pipeline (AODP), which identified all mutations distinguishing highly conserved DNA markers between close relatives. A generic bioinformatics pipeline was developed, including all steps used in this study from in silico evaluation of primers to accurate identification of short metabarcodes at the level of interest for defining phytopathogens. The results highlight the importance of primer selection, refDBs that are resolved to reflect phylogenetic relationships, and the use of AODP for improving the reliability of metabarcoding in phytopathogen biosurveillance.

Molecular diagnosis of dermatophyte isolates from canine and feline dermatophytosis in Northeast Iran.

BackgroundDermatophytes are the most common causes of cutaneous fungal diseases. Dermatophytosis is a common skin disorder in dogs and cats. Species identification of these fungi is important from a therapeutic and epidemiological aspect. Conventional methods used to identify dermatophyte species are often lengthy and may be inefficient in many circumstances. Recently broad varieties of several molecular DNA‐based techniques were successfully utilised for species detection of dermatophytes.ObjectivesThe aim of this study was to determine the molecular detection of dermatophyte isolates from canine and feline dermatophytosis in Mashhad, Iran.MethodsThirty dermatophytes isolated from dogs and cats with skin lesions and one standard strain of Microsporum canis were cultured onto Mycosel agar, and then internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using the universal fungal primers ITS1 and ITS4. PCR products were subjected to sequencing and sequence analysis.ResultsBased on the sequencing of the ITS1‐5.8S‐ITS2 region on all samples, all the studied strains were M. canis and their sexual stage (teleomorph) was Arthroderma otae.Conclusions Microsporum canis was the only species found among dogs and cats, and its high prevalence can increase the rate of transmission to humans. In practice, ITS‐PCR, with sequence analysis, is a useful and reliable method to identify and differentiate various pathogenic species, and it can be used in clinical and epidemiological fields, even for the rapid diagnosis of dermatophyte species that are closely interrelated.

Silver Decorated Myconanoparticles Control Growth and Biofilm Formation in Uropathogenic E. coli.

Nanotechnology involves the synthesis of nanoparticles that have been used in the therapeutic application for treating diseases. In this present study, we have adopted the synthesis of myconanoparticles from the extracellular extract of endophytic fungi Penicillium sclerotiorum (PsNps) and validated its antibacterial potential against antibiotic-resistant uropathogenic E. coli and ATCC (25,922) strain of Escherichia coli. Endophytic fungi were isolated from the healthy leaves of Tamarindus indica. The genomic DNA from endophytic fungi was isolated and the ITS region was amplified by polymerase chain reaction (PCR) using universal fungal primers ITS1 and ITS4 and sequenced for the identification of endophytic fungal isolates. Penicillium sclerotiorum extract was used for the synthesis of silver nanoparticles (PsNps) and was characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), zeta potential, FE-SEM, and Energy dispersive X-ray analysis (EDAX). Antibacterial activity of PsNps was tested against the antibiotic-resistant uropathogenic E. coli and ATCC (25,922) strain of E. coli. Further experiments were carried out to explore the potential of PsNps in regulating the CTX-M-15 gene. The antimicrobial activity showed that the PsNps inhibited growth, biofilm formation in both the strains of E. coli. The expression of the gene encoding CTX-M-15 was downregulated in a resistant strain of uropathogenic E. coli. Our results suggest that the PsNps could be used as an alternative source for antibiotics. Thus, further studies can be conducted to prove the in vivo potential of PsNps and can be formulated for commercialization.

First Report of Rhizoctonia solani AG-2-2 IIIB Causing Foliar Blight on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae), a perennial plant, is a traditional Chinese herb (known as baiji) used to treat hemorrhage, scalding injuries, gastric ulcers, pulmonary diseases, and inflammation (Zu et al. 2019). In May 2019, foliar blight symptoms were observed on approximately 25% of B. striata (cv. Guiji No.1) plants in three plantations (∼4.5 hectares in total) in Ziyuan County, Guangxi Province, China. Initial symptoms were light brown, irregular, water-soaked spots on the plant leaves. Several spots often merged, forming large, irregular, lesions that extended onto the stem after a week and led to leaf abscission, and even plant death. To determine the causal agent, 5-mm squares cut from the margin of 6 infected leaves were surface disinfected in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C (12-h light-dark cycle) for 3 days. The emerging hyphal tip of a single mycelium was transferred to PDA to obtain pure cultures of the isolates. Twenty isolates were obtained, and 10 isolates (50%) were initially white before turning light brown (∼4 days). Septate hyphae were 4.29 to 10.75 μm (average 6.42 μm) in diameter and branched at right angles with a constriction at the origin of the branch point. Staining with 1% safranin O and 3% KOH solution (Bandoni 1979) revealed multinucleated cells (3 to 9 nuclei per cell, n = 142). This morphology was typical of Rhizoctonia solani Kühn (Meyer et al. 1990). For species confirmation by molecular identification, three isolates (BJ101.6, BJ101.11, and BJ102.2) were cultured on PDA for 4 days, then DNA was extracted from the mycelium using the CTAB method (Guo et al. 2000), and the ribosomal ITS1-5.8S-ITS2 region was amplified by PCR using the universal fungal primers ITS1 and ITS4 (White et al. 1990). Internal transcribed spacer (ITS) sequences of strains BJ101.6, BJ101.11, and BJ102 (deposited in GenBank under accession nos MT406271, MT892815, and MT892814, respectively) had over 99% similarity with those of R. solani AG-2-2 IIIB in GenBank (accession nos JX913810 and AB054858) (Carling et al. 2002; Hong et al. 2012). Phylogenetic analysis using ITS sequences showed that the isolates clustered monophyletically with strains of R. solani AG-2-2 IIIB. The AG of the isolates was confirmed by their ability to grow well on PDA at 35°C, which separates AG-2-2 IIIB from AG-2-2 IV (Inokuti et al. 2019). Based on morphological characteristics and nucleotide sequence analysis, the isolates were identified as R. solani AG-2-2 IIIB. Pathogenicity was tested using 1.5-year-old B. striata (cv. Guiji No.1) plants grown in a perlite and peat moss mixture (1:3) in 7-cm pots. Healthy leaves on plants were inoculated with an aqueous suspension (approximately 1 × 105 hyphal fragments/mL, 100 μL) prepared from cultures of strains BJ101.6, BJ101.11, and BJ102.2, each isolate was inoculated onto three plants; three other plants with sterile water served as controls. All plants were enclosed in transparent plastic bags and incubated in a greenhouse at 28°C for 14 days (12-h photoperiod). Three days post-inoculation, leaves exposed to the mycelial fragments had symptoms similar to those originally observed in the field. No symptoms were detected on control plants. Experiments were replicated three times with similar results. To fulfill Koch's postulates, R. solani AG-2-2 IIIB was re-isolated on PDA from symptomatic leaves and confirmed by sequencing, whereas no fungus was isolated from the control plants. To our knowledge, this is the first report of R. solani AG-2-2 IIIB causing foliar blight on B. striata in China, and these findings will be useful for further control strategies and research.

A possible role for ticks in the transmission of Madurella mycetomatis in a mycetoma-endemic village in Sudan.

Currently there is a wide knowledge gap in our understanding of mycetoma epidemiological characteristics, including the infection route. A cross-sectional descriptive epidemiological study was carried out to determine the role of exposure to animals and insects such as ticks in the transmission of eumycetoma in two adjacent villages at eastern Sudan. Significant differences were found between the two villages in the level of contact and exposure to animals and ticks, the percentages of people bitten by ticks, participation in cleaning animal pens and knowledge of the medical importance of ticks. In the village with a high mycetoma prevalence rate, there were high infestation rates of ticks in domestic animals. Hyalomma and Rhipicephalus species were the most prevalent species in houses with mycetoma patients and together they constituted 83% of the total collection. Pool screening of vectors for the detection of Madurella mycetomatis recombinant RNA genes showed one positive pool from Rhipicephalus evertsi following amplification of the universal fungal primer and one positive sample from Hyalomma rufipes following the use of a specific primer. The findings indicate a possible role of ticks in the transmission of eumycetoma causative agents. However, further in-depth studies are needed to verify this.

Species diversity of Ganoderma causing foot rot of arecanut in southern dry tracts of Karnataka

Aim: The present study was undertaken to study the morphological and molecular diversity of Ganoderma spp. causing foot rot of arecanut in dry tracts of Southern Karnataka. Methodology: A total of 20 samples isolated from diseased areca palms in three districts of Karnataka were identified based on morphological and molecular characteristics. Qualitative data of cultural characteristics were transformed into code and a binary matrix was generated. Total genomic DNA was isolated and ITS region was amplified using universal primers ITS1 and ITS4. PCR amplicon was directly sequenced and phylogenetic analysis was carried out. Results: The dendrogram generated from the cultural morphological characteristics showed clear variations among Ganoderma isolates causing foot rot. DNA amplification of Ganoderma isolates with fungal universal primers (ITS1 and ITS4) was observed at 650 bp in all isolates tested. Taxonomic correlation of isolates upon NCBI web proved that the isolates were genetically related to Ganoderma spp. with 89-99.49 per cent identity and confirmed the taxonomic identity of isolates used in this study. Interpretation: Phylogenetic analysis of arecanut isolates of southern Karnataka are distinct as evidenced by forming separate cluster. Based on gene homology, G. ryvardenni and G. casuarinicola are new species reported as causal agent of foot rot in arecanut from Karnataka. The ITS gene sequences of four isolates viz., AG3 (MN 784436), AG4 (MN 784437), AG11 (MN 784438), and AG20 (MN 78449) were deposited in NCBI gene bank. Key words: Arecanut, Foot rot, Ganoderma, Phylogeny, Species diversity

Triplex Real-Time PCR Approach for the Detection of Crucial Fungal Berry Pathogens-Botrytis spp., Colletotrichum spp. and Verticillium spp.

Phytopathogens cause undeniably serious damage in agriculture by harming fruit cultivations and lowering harvest yields, which as a consequence substantially reduces food production efficiency. Fungi of the Botrytis, Colletotrichum and Verticillium genera are a main concern in berry production. However, no rapid detection method for detecting all of these pathogens simultaneously has been developed to date. Therefore, in this study, a multiplex real-time PCR assay for this purpose was established. Universal fungal primers for the D2 region of the large subunit ribosomal DNA and three multiplexable fluorogenic probes specific for the chosen fungi were designed and deployed. The triplex approach for the molecular detection of these fungi, which was developed in this study, allows for the rapid and effective detection of crucial berry pathogens, which contributes to a more rapid implementation of protective measures in plantations and a significant reduction in losses caused by fungal diseases.

PCR: An Effective, Faster and Relevant Method for the Detection of Clinical Isolates of Candida albicans

Background: Candida albicans is associated with infectious diseases with colonization, in both immunocompromised and immunocompetent patients. Antibiotic treatment can develop candidiasis. Objective: Evaluation of ITS 4 and ITS5 primer expression on clinical isolates of C. albicans. Methods: Fifty clinical isolates of C. albicans were collected from different sites viz. Catheter Tip (CT), High Vaginal Swab (HVS) and Urine (U). Results: Using the universal fungal primers ITS4 and ITS5, PCR produced a single monomorphic amplification at 520 bp indicating that the strains are of same genetic identity. Conclusion: This is an effective, faster and relevant method for the detection and characterization of clinically isolated strains of C. albicans.

The mycobiomes and bacteriomes of sputum, saliva, and home dust.

Respiratory microbiome is an understudied area of research compared to other microbiomes of the human body. The respiratory tract is exposed to an array of environmental pollutants, including microbes. Yet, we know very little about the relationship between environmental and respiratory microbiome. The primary aim of our study was to compare the mycobiomes and bacteriomes between three sample types from the same participants, including home dust, saliva, and sputum. Samples were collected from 40 adolescents in a longitudinal cohort. We analyzed the samples using 16s bacterial rDNA and ITS fungal rDNA gene sequencing, as well as quantitative PCR with universal fungal and bacterial primers. Results showed that home dust had the greatest alpha diversity between the three sample types for both bacteria and fungi. Dust had the highest total fungal load and the lowest total bacterial load. Sputum had greater bacterial diversity than saliva, but saliva had greater fungal diversity than sputum. The distribution of major bacterial phyla differed between all sample types. However, the distribution of major fungal classes differed only between sputum and saliva. Future research should examine the biological significance of the taxa found in each sample type based on microbial ecology and associations with health effects.

First Report of Ceratobasidium sp. Causing Root Rot of Garlic in China

Garlic (Allium sativum L.) is an important cash crop in China. In the spring of 2018, stunted and chlorotic plants in some garlic fields were observed in Jinxiang, Shandong, and Feng Counties, Jiangsu Province, in China. The growth of symptomatic plants was weaker, with affected plants showing signs of apical chlorosis, wilting, and dieback. In the aerial part of the plant, the symptoms of root rot disease resembled nutrient deficiency or drought stress. The roots were shriveled and rotted, with reduced root density. According to survey, the incidence of root rot in each garlic planting area is as high as 35 to 40%, and once the disease occurs, the commodity value of garlic will be reduced. Generally speaking, fungal diseases are among the main biotic factors affecting garlic yield, particularly those caused by species of the genera Fusarium, Phytophthora, and Pythium. In order to identify the causal agent of this disease, symptomatic plants were collected from several regions of the main garlic producing areas in China, including Jinxiang County, Jiaxiang County in Shandong Province, and Fengxian County in Jiangsu Province. The garlic variety planted is Jinxiang white garlic, which is a local garlic variety. Samples of rotten roots were washed in tap water, surface sterilized in 1.5% sodium hypochlorite for 1 min followed by 75% ethanol for 30 s, rinsed with sterilized distilled water three times, and blotted dry on sterile filter paper. Pieces of infected root tissues (2 to 3 mm) were placed on potato dextrose agar (PDA) and kept at 28°C for 7 days. After the colonies were established on PDA, the fungal strains were purified by the hyphal-tip method. Five fungal isolates with similar morphology to each other were obtained, and isolate DS54-3 was chosen for the following experiment. The fungal colony grew within 5 days on PDA containing a full culture medium. Fungal colonies were white initially and then turned brown, and septate hyphae were 3.7 to 4.3 μm in diameter and branched at right angles with a constriction at the origin of the branch point. These characteristics are typical of the genus Rhizoctonia. Binucleate cells from five isolates were observed using a lactophenol aniline blue solution stain and matched Ceratobasidium morphological descriptions (Chen et al. 1985). The morphological characteristics of isolate DS54-3 were consistent with Ceratobasidium sp. (Sneh et al. 1991). Mycelia from five isolates grown on PDA were used for DNA extraction. The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (White et al. 1990). The purified products were separately sequenced in both directions using the same primer pair (Meza-Moller et al. 2014). ITS sequence analysis (GenBank accession no. MK733974) resulted in a 100% match for one accession of Ceratobasidium sp. AG-A (MF070683.1) by BLAST in the NCBI nucleotide database. The pathogenicity of isolate DS54-3 was then tested. For each conidial suspension,12 healthy 10-day-old garlic plants (about 5 cm) were used to conduct pathogenicity tests by wound inoculation, and the garlic varieties were the same as those collected in the field. The healthy garlic plants were inoculated with 5, 10, 20, 50, and 100 ml of conidial suspension (2.95 × 10⁷ conidia/ml) by the root irrigation method. Twenty plants were inoculated with the same volume of sterile water as a control for each conidial suspension. After 20 days, the leaves of all garlic seedlings inoculated with conidial suspension gradually turned yellow, similar to the symptoms observed during natural infection. After 40 days, the root system showed typical hollow and rotten symptoms, and eventually the plants withered and died, whereas the control plants remained healthy. The same colonial fungus was reisolated, thus fulfilling Koch’s postulates. This is the first report of Ceratobasidium sp. causing root rot disease of garlic in China.

Isolation of Different Species of Candida in Patients with Vulvovaginal Candidiasis from Damavand, Iran

Background: One of the most common infections in women, especially during the reproductive age, is vulvovaginal candidiasis, which can cause physical and psychological distress for patients. According to different reports, Candida albicans is the main etiological agent of this infection. However, non-Candida albicans species have arisen recently. Therefore, it seems that in VVC, the pattern of Candida species, have changed. Objectives: In the current study, the researchers determined the frequency of different species of Candida in the Iranian female population. Methods: Five hundred and fifty-nine patients suspected of VVC, participated in this study. Wet swabs were used for collecting the samples. Vaginal swabs were examined microscopically and treated for fungal culture. Candida species identification was done by polymerase chain reaction (PCR)-Sequencing with universal fungal primers (ITS1 and ITS4). Results: Out of 559 patients, 46 (8.2%) samples were positive for VVC. The age group of 30 to 39 years old with 20 (44.7%) patients had the highest frequency. Itching concomitant with abnormal discharge and vaginal irritation (59.6%) were the most prevalent symptoms in the patients. The most frequent identified species of Candida were Candida albicans (67.7%), Candida glabrata (25.8%), and Candida kefyr (3.2%), respectively. In addition, only one (2.1%) case showed recurrent vulvovaginal candidiasis (RVVC). Conclusions: The results of the present study demonstrated that in patients with VVC, Candida albicans species are more frequent than other candida species. In order to achieve appropriate treatment, clinical and laboratory findings must be considered together.

Assessment of Soil Fungal Diversity at Different Depth in Haibat Sultan Mountain by PCR-Cloning-Sequencing Methods

Understanding the fungal community structure of soil is important for optimizing their role as decomposers in the soil food web. In order to explore the fungal composition and diversity in Haibat Sultan Mountain soil, PCR used based cloning and sequencing of Internal Transcribed Spacer (ITS) of genome. Soil samples were collected from different depth points (1, 5 to 10 cm depth) and fungal universal primers targeting ITS2 region of the fungal genome were used to assess their diversity in soil samples. More than 1400 fungal clones were sequenced and led to detect total of 89 OTUs in all samples. The majority of the Operational Taxonomic Units (OTUs) belong to the Ascomycota (67.4%), the second most constituents of soil samples were Basidiomycota with (28.1%), and few members of Zygomycota were detected. Concluded that fungal richness and diversity were abundant at the depth of 1 cm and reached their peak at the depth of 5 cm, while at the depth of 10 cm, the fungal communities decreased. The highest Chao1 and Shannon value were obtained at the soil depth of 5 cm followed by soil depth 1cm, whereas the lowest value was detected in soil depth 10 cm. Results highlight the variations of fungal diversity at different depth points. Thus, further studies are needed to expand knowledge about fungi communities in soil through using high-throughput sequencing to improve detection fungi in soil

Assessing the abundance of fungal populations in a full-scale membrane bioreactor (MBR) treating urban wastewater by using quantitative PCR (qPCR)

The abundance of fungi in a full-scale membrane bioreactor (MBR) treating urban wastewater and experiencing seasonal foaming was assessed by quantitative PCR (qPCR), comparing three different sets of widely used universal fungal primers targeting the gene encoding the small ribosomal subunit RNA, 18S-rDNA, (primers NS1-Fung and FungiQuant) or the internal transcribed spacer ITS2 (primers ITS3-ITS4). Fungi were a numerically important fraction of the MBR microbiota (≥106 18S-rDNA copies/L activated sludge), and occurred both in the aerated and anoxic bioreactors. The numbers of copies of fungal markers/L activated sludge calculated using the NS1-Fung or ITS3-ITS4 primer sets were up to 2 orders of magnitude higher than the quantifications based on the FungiQuant primers. Fungal 18S-rDNA counts derived from the FungiQuant primers decreased significantly during cold seasons, concurring with foaming episodes in the MBR. Redundancy analysis corroborated that temperature was the main factor driving fungi abundance, which was also favored by longer solid retention time (SRT), lower chemical oxygen demand/biochemical oxygen demand at 5 days (COD/BOD5) of influent water, and lower biomass accumulation in the MBR.