Total Fungal DNA Research Articles

Composition and dynamics of intestinal fungi during the postnatal 2 months of very low birth weight infants

It has been found that intestinal fungi play a role in the composition of the intestinal microecology and in the formation and development of the immunity during childhood. We investigated the gut fungi composition of preterm infants to analysis composition and dynamics of intestinal fungi during the postnatal 2 months of very low birth weight infants. We collected feces from 34 very low birth weight infants (VLBWI) and 28 preterm infants with birth weight >1500 g. We extracted total fungal DNA from feces and analyzed the composition of gut fungus through ITS sequencing. The fungal detectable rate in the experimental group peaked on day 3 (85.19%), then gradually decreased and started to show an increasing trend again by day 28. There were significant differences in the alpha diversity of intestinal fungus between VLBWI and controls, and the VLBWI had its own characteristics at different time points in richness and diversity. A total of 10 phylums and 342 genera were identified in all VLBWI samples. The dominant fungal phylum of the VLBWI group is Ascomycota (50.3%)and Basidiomycota (48.8%). The functional metabolic activity of the experimental group was lower than that of the control group. Conclusion: The composition and abundance of VLBWI intestinal fungal showed several alterations during the first 2 months of life. The prediction of gut microbiota function suggests that intestinal metabolic function may be altered in VLBWI.What is Known:• A limited number of studies has been found that symbiont fungi may be able to calibrate host immunological responses, promote development of peripheral lymphoid organs, promote T cell responses, and even may be associated with the development of certain diseases, such as inflammatory bowel disease (IBD), NEC, and allergic diseases. However, previous studies on intestinal microecology have mainly focused on adults while neglecting the role of fungi in the gut of children due to the much lower abundance of intestinal fungi than bacteria, limitations of techniques for detecting fungi, the difficulty of obtaining samples, and the absence of largescale reference databases.What is New:• In recent years, the discovery and development of fungal detection technologies such as 18s rDNA sequencing technology, Internal Transcribed Spacer(ITS), and DNA fingerprinting technology have further broadened the perspective on the impact of intestinal fungal exposure in early life.

Evolution of fungal and non-fungal eukaryotic communities in response to thermophilic co-composting of various nitrogen-rich green feedstocks.

Thermophilic composting is a promising soil and waste management approach involving diverse micro and macro-organisms, including eukaryotes. Due to sub-optimal amounts of nutrients in manure, supplemental feedstock materials such as Lantana camara, and Tithonia diversifolia twigs are used in composting. These materials have, however, been reported to have antimicrobial activity in in-vitro experiments. Furthermore, the phytochemical analysis has shown differences in their complexities, thus possibly requiring various periods to break down. Therefore, it is necessary to understand these materials' influence on the biological and physical-chemical stability of compost. Most compost microbiome studies have been bacterial-centric, leaving out eukaryotes despite their critical role in the environment. Here, the influence of different green feedstock on the fungal and non-fungal eukaryotic community structure in a thermophilic compost environment was examined. Total community fungal and non-fungal eukaryotic DNA was recovered from triplicate compost samples of four experimental regimes. Sequencing for fungal ITS and non-fungal eukaryotes; 18S rDNA was done under the Illumina Miseq platform, and bioinformatics analysis was done using Divisive Amplicon Denoising Algorithm version 2 workflow in R version 4.1. Samples of mixed compost and composting day 84 recorded significantly (P<0.05) higher overall fungal populations, while Lantana-based compost and composting day 84 revealed the highest fungal community diversity. Non-fungal eukaryotic richness was significantly (P< 0.05) more abundant in Tithonia-based compost and composting day 21. The most diverse non-fungal eukaryotic biome was in the Tithonia-based compost and composting day 84. Sordariomycetes and Holozoa were the most contributors to the fungal and non-fungal community interactions in the compost environment, respectively. The findings of this study unravel the inherent influence of diverse composting materials and days on the eukaryotic community structure and compost's biological and chemical stability.

Gender Effects of Dioecious Plant Populus cathayana on Fungal Community and Mycorrhizal Distribution at Different Arid Zones in Qinghai, China

Dioecious plants have a wide distribution in nature and gender effect may cause significant alterations in rhizosphere fungal community and soil properties. However, little is known regarding changes in response to dioecious plants. This study aimed to investigate the effects that the dioecious plant, Populus cathayana, and regions of different arid levels have on the fungal community, mycorrhizal distribution, soil enzymatic activities, and nutrient contents. This study characterized fungal and soil factors from the rhizosphere of the dioecious plant Populus cathayana located in the semi-humid regions (Chengguan), semi-arid regions (Sining, Haiyan) and arid regions (Ulan, Chaka). Rhizosphere soil was collected from each site and gender, and the total fungal genomic DNA was extracted. DNA amplicons from fungal ITS region were generated and subjected to Illumina Miseq sequencing. A total of 5 phyla, 28 classes, 92 orders, 170 families, and 380 genuses were observed. AMF distribution peaked at Chaka, which did not conform to the trend. Gender had significant effects on fungal communities: there were obvious differences in fungal OTUs between genders. Alpha diversity raised at first and then decreased. RDA results showed available P, available K, pH, ALP activity, ammonium N, EC, water content and catalase activity were the key contributors in sample areas. Our results suggested potential interaction effects between plant gender and fungal community.

Composition of Rhizosphere Microbial Communities Associated With Healthy and Verticillium Wilt Diseased Cotton Plants.

Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V. dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community.

Microdochium majus and other fungal pathogens associated with reduced gluten quality in wheat grain

The bread-making quality of wheat depends on the viscoelastic properties of the dough in which gluten proteins play an important role. The quality of gluten proteins is influenced by the genetics of the different wheat varieties and environmental factors. Occasionally, a near complete loss of gluten strength, measured as the maximum resistance towards stretching (Rmax), is observed in grain lots of Norwegian wheat. It is hypothesized that the loss of gluten quality is caused by degradation of gluten proteins by fungal proteases. To identify fungi associated with loss of gluten strength, samples from a selection of wheat grain lots with weak gluten (n = 10, Rmax < 0.3 N) and strong gluten (n = 10, Rmax ≥ 0.6 N) was analyzed for the abundance of fungal operational taxonomic units (OTUs) using DNA metabarcoding of the nuclear ribosomal Internal Transcribed Spacer (ITS) region ITS1. The DNA quantities for a selection of fungal pathogens of wheat, and the total amount of fungal DNA, were analyzed by quantitative PCR (qPCR). The mean level of total fungal DNA was higher in grain samples with weak gluten compared to grain samples with strong gluten. Heightened quantities of DNA from fungi within the Fusarium Head Blight (FHB) complex, i.e. Fusarium avenaceum, Fusarium graminearum, Microdochium majus, and Microdochium nivale, were observed in grain samples with weak gluten compared to those with strong gluten. Microdochium majus was the dominant fungus in the samples with weak gluten. Stepwise regression modeling based on different wheat quality parameters, qPCR data, and the 35 most common OTUs revealed a significant negative association between gluten strength and three OTUs, of which the OTU identified as M. majus was the most abundant. The same analysis also revealed a significant negative relationship between gluten strength and F. avenaceum detected by qPCR, although the DNA levels of this fungus were low compared to those of M. majus. In vitro growth rate studies of a selection of FHB species showed that all the tested isolates were able to grow with gluten as a sole nitrogen source. In addition, proteins secreted by these fungi in liquid cultures were able to hydrolyze gluten substrate proteins in zymograms, confirming their capacity to secrete gluten-degrading proteases. The identification of fungi with potential to influence gluten quality can enable the development of strategies to minimize future problems with gluten strength in food-grade wheat.

Diversity, Bacterial Symbionts, and Antimicrobial Potential of Termite-Associated Fungi.

The phylogenetic diversity of fungi isolated from the Odontotermes formosanus was investigated by dilution-plate method, combined with morphological characteristics and 5.8S rDNA sequencing. Thirty-nine fungi were isolated and purified from O. formosanus, which were belonging to two phyla and four classes (Sordariomycetes, Dothideomycetes, Eurotiomycetes, Agaricomycetes). Furthermore, nine bacterial 16S rRNA sequences were obtained from total fungal genomic DNA. All bacterial symbionts were segmented into four genera: Bacillus, Methylobacterium, Paenibacillus, and Trabulsiella. The antimicrobial activities of all endophytic fungi extracts were tested by using the filter paper method against Escherichia coli (ATCC 8739), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), and Canidia albicans (ATCC 10231). The results exhibited that 25 extracts (64%) exhibited antibacterial activity against at least one of the tested bacterial strains. Furthermore, the secondary metabolites 1 [5-hydroxyramulosin (1a):biatriosporin M (1b) = 2:1] from the Pleosporales sp. BYCDW4 exhibited potent antimicrobial activities against E. coli, C. albicans, B. subtilis, and S. aureus with the inhibition zone diameter (IZD) of 13.67, 14.33, 12.17, and 11.33 mm, respectively, which were comparable with those of the positive control. 1-(2,5-Dihydroxyphenyl)-3-hydroxybutan-1-one (2) from the Microdiplodia sp. BYCDW8 showed medium inhibitory activities against B. subtilis and S. aureus, with the IZD range of 8.32–9.13 mm. In conclusion, the study showed the diversity of insect symbionts could be expected to develop the resource of new species and antibiotics.

Morphology and quantification of fungal growth in residential dust and carpets

Mold growth indoors is associated with negative human health effects, and this growth is limited by moisture availability. Dust deposited in carpet is an important source of human exposure due to potential elevated resuspension compared to hard floors. However, we need an improved understanding of fungal growth in dust and carpet to better estimate human exposure. The goal of this study was to compare fungal growth quantity and morphology in residential carpet under different environmental conditions, including equilibrium relative humidity (ERH) (50%, 85%, 90%, 95%, 100%), carpet fiber material (nylon, olefin, wool) and presence/absence of dust. We analyzed incubated carpet and dust samples from three Ohio homes for total fungal DNA, fungal allergen Alt a 1, and fungal morphology. Dust presence and elevated ERH (≥85%) were the most important variables that increased fungal growth. Elevated ERH increased mean fungal DNA concentration (P < 0.0001), for instance by approximately 1000 times at 100% compared to 50% ERH after two weeks. Microscopy also revealed more fungal growth at higher ERH. Fungal concentrations were up to 100 times higher in samples containing house dust compared to no dust. For fiber type, olefin had the least total fungal growth, and nylon had the most total fungi and A. alternata growth in unaltered dust. Increased ERH conditions were associated with increased Alt a 1 allergen concentration. The results of this study demonstrate that ERH, presence/absence of house dust, and carpet fiber type influence fungal growth and allergen production in residential carpet, which has implications for human exposure.

Quantitative filter forensics with residential HVAC filters to assess indoor concentrations.

Analysis of the dust from heating, ventilation, and air conditioning (HVAC) filters is a promising long-term sampling method to characterize airborne particle-bound contaminants. This filter forensics (FF) approach provides valuable insights about differences between buildings, but does not allow for an estimation of indoor concentrations. In this investigation, FF is extended to quantitative filter forensics (QFF) by using measurements of the volume of air that passes through the filter and the filter efficiency, to assess the integrated average airborne concentrations of total fungal and bacterial DNA, 36 fungal species, endotoxins, phthalates, and organophosphate esters (OPEs) based on dust extracted from HVAC filters. Filters were collected from 59 homes located in central Texas, USA, after 1month of deployment in each summer and winter. Results showed considerable differences in the concentrations of airborne particle-bound contaminants in studied homes. The airborne concentrations for most of the analytes are comparable with those reported in the literature. In this sample of homes, the HVAC characterization measurements varied much less between homes than the variation in the filter dust concentration of each analyte, suggesting that even in the absence of HVAC data, FF can provide insight about concentration differences for homes with similar HVAC systems.

Ocular symptoms and tear film break up time (BUT) among junior high school students in Penang, Malaysia - Associations with fungal DNA in school dust.

There are few studies on ocular effects of indoor mould exposure in schools, especially in the tropics OBJECTIVE: To study associations between eye symptoms and tear film break up time (BUT) in students and demographic data and fungal DNA in schools. A school environment study was performed among randomly selected students in eight randomly selected secondary schools in Penang, Malaysia. Information on eye symptoms and demographic data was collected by a standardised questionnaire. BUT was measured by two methods, self-reported BUT (SBUT) and by the non-invasive Tearscope (NIBUT). Dust was collected by vacuuming in 32 classrooms and analysed for five fungal DNA sequences. Geometric mean (GM) for total fungal DNA was 7.31*104 target copies per gram dust and for Aspergillus/Penicillium DNA 3.34*104 target copies per gram dust. Linear mixed models and 3-level multiple logistic regression were applied adjusting for demographic factors. A total of 368 students (58%) participated and 17.4% reported weekly eye symptoms the last 3 months. The median SBUT and TBUT were 15 and 12s, respectively. Students wearing glasses (OR 2.41, p=0.01) and with a history of atopy (OR=2.67; p=0.008) had more eye symptoms. Girls had less eye symptoms than boys (OR=0.34; p=0.006) Indoor carbon dioxide in the classrooms was low (range 380-720ppm), temperature was 25-30°C and relative air humidity 70-88%. Total fungal DNA in vacuumed dust was associated with shorter SBUT (4s shorter per 105 target copies per gram dust; p=0.04) and NIBUT (4s shorter per 105 target copies per gram dust; p<0.001). Aspergillus/Penicillium DNA was associated with shorter NIBUT (5s shorter per 105 target copies per gram dust; p=0.01). Fungal contamination in schools in a tropical country can be a risk factor for impaired tear film stability among students.

Rhinitis, Ocular, Throat and Dermal Symptoms, Headache and Tiredness among Students in Schools from Johor Bahru, Malaysia: Associations with Fungal DNA and Mycotoxins in Classroom Dust.

There are few studies on rhinitis and sick building syndrome (SBS) among students in tropical countries. We studied associations between levels of five fungal DNA sequences, two mycotoxins (sterigmatocystin and verrucarol) and cat allergen (Fel d 1) levels in schools and rhinitis and other weekly SBS symptoms in the students. Fungal DNA was measured by quantitative PCR and cat allergen by ELISA. Pupils (N = 462) from eight randomly selected schools in Johor Bahru, Malaysia participated (96%). Dust samples were collected by cotton swabs and Petri dishes exposed for one week. None of the schools had a mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures and indoor CO2 levels were low (mean 492 ppm; range 380–690 ppm). Weekly nasal symptoms (rhinitis) (18.8%), ocular (11.6%), throat (11.1%), dermal symptoms, headache (20.6%) and tiredness (22.1%) were common. Total fungal DNA in swab samples was associated with rhinitis (p = 0.02), ocular symptoms (p = 0.009) and tiredness (p = 0.001). There were positive associations between Aspergillus versicolor DNA in Petri dish samples, ocular symptoms (p = 0.02) and tiredness (p = 0.001). The level of the mycotoxin verrucarol (produced by Stachybotrys chartarum) in swab samples was positively associated with tiredness (p = 0.04). Streptomyces DNA in swab samples (p = 0.03) and Petri dish samples (p = 0.03) were negatively associated with tiredness. In conclusion, total fungal contamination, measured as total fungal DNA) in the classrooms, Aspergillus versicolor and verrucarol can be risk factors for rhinitis and SBS symptoms among students in the tropical country Malaysia.

Fungal DNA in dust in Swedish day care centres: associations with respiratory symptoms, fractional exhaled nitrogen oxide (FeNO) and C-reactive protein (CRP) in serum among day care centre staff.

To study associations between fungal DNA in day care centres, fractional exhaled nitric oxide (FeNO) and inflammatory markers in day care centre staff. Totally, 62 staff (90 %) from five day care centres in Sweden participated. All were females. Settled dust was collected and analysed for five sequences of fungal DNA by quantitative PCR. Levels of FeNO (NIOX MINO 50 ml/min) and serum levels of eosinophilic cationic protein, myeloperoxidase (MPO) and high-sensitivity C-reactive protein in blood (HsCRP) were measured. Dynamic spirometry was performed, and dyspnoea was measured. Biomarkers and dyspnoea ratings were log-transformed, and associations were analysed by linear mixed models, adjusting for age, atopy, smoking, body mass index (BMI), ETS and dampness/mould at home. Geometric mean (GM) for FeNO was 15.3 ppb, 6% were smokers, 14% were obese, 31% were overweight and 18% had atopy. GM concentration was 2.16 × 10(5) cell equivalents (CE)/g for total fungal DNA, 2310 CE/g for Aspergillus/penicillium (Asp/Pen) DNA, 17 CE/g for Aspergillus versicolor DNA and 14 CE/g dust for Streptomyces DNA. FeNO was associated with total fungal DNA (p = 0.004), Asp/Pen DNA (p = 0.005) and Streptomyces DNA (p = 0.03). HsCRP was associated with total fungal DNA (p = 0.03) and BMI (p = 0.001). Dyspnoea was associated with Asp/Pen DNA (p = 0.04). Subjects with ETS at home had lower lung function (FEV1) (p = 0.03), and those with dampness/mould at home had lower MPO (p = 0.03). Fungal contamination in day care centres, measured as fungal DNA, can be a risk factor for airway inflammation, and CRP is associated with BMI.

First Report of Powdery Mildew of Platanus × acerifolia and P. occidentalis Caused by Erysiphe platani in Greece.

London planetrees (Platanus × acerifolia, syn. P. × hispanica), American sycamores (P. occidentalis), and oriental planes (P. orientalis) are widely planted as urban shade trees throughout Greece and many other countries. In June 2012, typical symptoms of a powdery mildew were detected on all sycamores (10 trees) along a central avenue of Heraklion (Crete, Greece), with the disease affecting approximately 80% of the leaves of all infected trees. In August 2013, similar symptoms were observed on 20% of the leaves of all three London planes in a small grove in the Vrysses area of Lasithi (Crete, Greece). In both cases, the disease was severe, with white superficial colonies developing amphigenously on leaves, twigs, floral peduncles, inflorescences, and fruits. The colonies were initially distinct and circular but gradually enlarged and often coalesced to cover the entire leaf blade. Young leaves appeared curled and chlorotic, occasionally leading to defoliation. For the morphological description of the pathogen, samples from seven infected P. occidentalis and three P. × acerifolia trees were microscopically characterized. In all samples, the pathogen's mycelium was branched, septate, and hyaline, with lobed appressoria; conidiophores were erect, cylindrical, unbranched, and consisted of three to four (to five) cells; and conidia were single or in short chains (two to four), ellipsoid or doliiform, with a truncated base and rounded apex. Their dimensions were 24.3 to 48.6 × 15.8 to 27.9 μm (averaging 39.2 × 21.2 μm; n = 100), and their surfaces appeared reticulate. The teleomorph was never observed. Total fungal DNA was extracted from conidia harvested from affected leaves of one infected plant of each of P. occidentalis and P. × acerifolia planes, and the ITS1-5.8S-ITS2 region was PCR-amplified with universal primers 18S-ITS1 and 28S-ITS2 (2) and sequenced (GenBank Accession Nos. KM068123 and KM068124, respectively). A BLASTn search of GenBank revealed 100% identity of both samples to Erysiphe platani strains described on P. orientalis in Greece (JQ365943) and P. occidentalis in Brazil (KF499270). Based on the morphological and molecular analyses, the pathogen was identified as E. platani (Howe) U. Braun & S. Takam. (formerly known as Microsphaera platani Howe) (1). To prove pathogenicity and fulfill Koch's postulates, 10 1-year-old seedlings of each of P. occidentalis and P. × acerifolia hosts were artificially inoculated with conidia obtained from naturally infected plants of the corresponding species, with two methods: (i) five plants of each host were dusted with conidia from diseased leaves, and (ii) the remaining five seedlings of each plane were sprayed with a conidial suspension of the fungus (107 conidia ml-1), while five additional control plants of each species were treated only with sterile distilled water. All plants were maintained in the greenhouse at 25 ± 3°C, with 90% humidity. Powdery mildew symptoms, which appeared 9 and 15 days after inoculation on all dusted and sprayed plants, respectively, were similar to those observed on naturally infected trees, whereas no symptoms were observed on control plants. Although E. platani is known to infect plane species in several parts of the world (1), including oriental planes (P. orientalis and P. orientalis var. cretica) in Greece (3), this is the first report of E. platani causing disease of P. occidentalis and P. × acerifolia in Greece, underlining the need for appropriate control measures to prevent significant losses to the local ornamental industry.

Fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique

To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota. According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. PCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n=24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69log10 CFUg(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ±0.07log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51log10 CFUg(-1) culturable fungi, while there was <2.83log10 CFUg(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.

First Report of New Postharvest Rot in Ginger Rhizome by Aspergillus parvisclerotigenus in Pakistan.

Ginger (Zingiber officinale) rhizome is widely used in Pakistan as a spice. During the summer of 2013, several ginger sellers in a local vegetable market of Lahore, Pakistan, reported a green powdery mass of an unidentified pathogen that rotted a considerable quantity of ginger in its packaging. As far as we know, this disease was limited to stored rhizomes and not reported in fields. A survey was conducted in August to September of 2013 in three different vegetable markets in Lahore to collect infected samples. From each of three survey points from individual markets, 20 bags (10 kg each) were selected randomly. Average incidence of decay (by weight) was found to be 45%. Initial symptoms appeared as discoloration, soft and slippery skin with abundant green sporulation. Ten samples (rhizomes) from each market were brought to the laboratory for further studies. Isolation of the causal agent was carried out on two growth media: malt extract agar (MEA) and Czapek Dox agar (CZA). Inoculation was carried out by direct transfer of visible green spores as well as transferring a small fragment of surface sterilized infected rhizome to the media. Inoculated media plates were incubated at 25°C for 3 to 4 days. Emerging fungal colonies were sub-cultured to get pure cultures. The fungal colony was powdery, green, 3.5 to 4 cm in diameter, and without zonation after 7 days of incubation. Sclerotia were brown to black and globose. Conidial heads were columnar and biseriate, occasionally unseriate. Conidiophores were 1 to 2.5 mm long. Vesicles were sub-globose to globose and 25 to 30 μm wide. Metulae were 12 to 18 μm high and phialides were 6 to 12 μm. Conidia were globose to sub-globose, green, and 4 to 5 μm in diameter. Based on morphology, the fungus was identified as Aspergillus parvisclerotigenus (1). The identity of the pathogen was confirmed by ITS sequence analysis of two different isolates. For this, ITS1-5.8S-ITS2 nucleotide sequence of ~560 bp was amplified using total fungal genomic DNA as a template and ITS1 forward (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 reverse primer (5'-TCCTCCGCTTATTGATATGC-3') (2). Sequences from both isolates were 100% similar with each other. A BLAST search showed that this sequence had 99% homology with that A. parvisclerotigenus CBS 121.62 (EF409240.1). A culture of the fungus was deposited in First Fungal Culture Bank of Pakistan (FCBP1352) and the nucleotide sequence of ITS region to GenBank (KJ445022). For completion of Koch's postulates, a spore suspension (105 spores/ml) from a 1-week-old culture was prepared. Ten surface-disinfested, air-dried ginger rhizomes were placed on sterilized wet blotting papers in a glass beaker and inoculated by spore suspension using a hand sprayer. Similarly, 10 control rhizomes were sprayed with sterile distilled water. Rhizomes were incubated at 25°C for 7 days. The experiment was replicated three times. The same symptoms noticed in the vegetable markets were observed in 80% of the inoculated rhizomes while control rhizomes remained healthy. Re-isolation of the pathogen from symptomatic rhizomes fulfilled Koch's postulates. Poor hygiene is thought to be the main cause of rotting; therefore, this disease is not a threat to ginger if stored properly. To our knowledge, this is the first report of postharvest ginger rhizome rot from Pakistan caused by A. parvisclerotigenus.

Associations between Quantitative Measures of Fungi in Home Floor Dust and Lung Function among Older Adults with Chronic Respiratory Disease: A Pilot Study

Objective. Many fungi may cause allergic reactions and increase asthma symptoms prevalence and severity. One susceptible, vulnerable population subgroup of increasing size in industrialized countries and of public health concern who spends the majority of their time at home is older adults. Older adults diagnosed with chronic obstructive pulmonary disease (COPD) are at risk of exposure to fungi. Currently, species identification is based on observation of microscopic and macroscopic morphologies, which may underestimate concentrations compared to quantitative polymerase chain reaction (qPCR)-based measurements which are toxicologically more relevant to exposure science. Methods. This article analyzes quantitative indoor exposure data on fungi in floor dust (cells/cm2 floor) by real-time qPCR-based detection with quantitative outcome data via field spirometry in a pilot community-based study in Visalia, Tulare County, California, between July 2009 and January 2010. Subjects (n = 9, five females, four males) were Caucasian, English-speaking, nonsmoking older adults with doctor-diagnosed asthma and/or COPD. Results. While certain results did not reach statistical significance (p ≤ .10) due to sample size—overall and by gender—we found consistent trends and statistically significant associations for total fungal DNA (summer data) with forced vital capacity and forced expiratory volume in 1 second. Conclusions. The results of this pilot study are novel and suggest adverse effects of exposure inside homes to certain fungal species. This pilot study supports the need for larger prospective epidemiologic studies of older adults with asthma and/or chronic bronchitis based on quantitative environmental and clinical measures.

Total viable molds and fungal DNA in classrooms and association with respiratory health and pulmonary function of European schoolchildren

Indoor molds are associated with adverse respiratory effects in children. Although schools are important exposure sources of molds, objective measurements were more often taken in homes. Our aim was to assess indoor molds in schools and related effects on schoolchildren health. The Health Effects of the School Environment study (HESE) included 21 schools (46 classrooms) in Italy, Denmark, Sweden, Norway, and France and 654 schoolchildren (mean age 10 yr). Information on schoolchildren was collected by standardized questionnaires. Measurements of total viable molds (VM, colony-forming units, cfu/m(3)) and total/specific fungal DNA (cell equivalents, CE/g dust) were taken inside all classrooms in the cold season during normal activities, using the same standardized methodology. Pulmonary function tests were performed on 244 pupils. VM (mean, 320,cfu/m(3)) and total fungal DNA (geometric mean, 2.2 × 10(5) ± 2.1 CE/g dust) were detectable in all classrooms. The levels were significantly higher in buildings with mold/dampness problems. VM, but not fungal DNA, were inversely related to ventilation rate. VM exceeded the maximum standard of 300 cfu/m(3) in 33% of the classrooms. In the past 12 months, dry cough at night (34%) and rhinitis (32%) were the mostly reported. Children exposed to VM levels ≥ 300 cfu/m(3), compared with those exposed to lower levels, showed higher risk for past year dry cough at night (odds ratio, OR: 3.10, 95% confidence interval, CI: 1.61-5.98) and rhinitis (OR: 2.86, 95% CI: 1.65-4.95), as well as for persistent cough (OR: 3.79, 95% CI: 2.40-5.60). Aspergillus/Penicillium DNA was significantly positively associated with wheeze, and Aspergillus versicolor DNA with wheeze, rhinitis, and cough. There were significant inverse associations of Aspergillus versicolor DNA with forced vitality capacity (FVC) and Streptomyces DNA with both FEV(1) and FVC. In conclusion, indoor VM and fungal DNA were commonly found in monitored European schools and adversely related to respiratory health. Schools should be routinely tested through both culturable and non-culturable methods for global indoor molds' evaluation.

A longitudinal study of sick building syndrome among pupils in relation to microbial components in dust in schools in China

There are few longitudinal studies on sick building syndrome (SBS), which include ocular, nasal, throat, and dermal symptoms, headache, and fatigue. We studied the associations between selected microbial components, fungal DNA, furry pet allergens, and incidence and remission of SBS symptoms in schools in Taiyuan, China. The study was based on a two-year prospective analysis in pupils (N=1143) in a random sample of schools in China. Settled dust in the classrooms was collected by vacuum cleaning and analyzed for lipopolysaccharide (LPS), muramic acid (MuA), and ergosterol (Erg). Airborne dust was collected in Petri dishes and analyzed for cat and dog allergens and fungal DNA. The relationship between the concentration of allergens and microbial compounds and new onset of SBS was analyzed by multi-level logistic regression. The prevalence of mucosal and general symptoms was 33% and 28%, respectively, at baseline, and increased during follow-up. At baseline, 27% reported at least one symptom that improved when away from school (school-related symptoms). New onset of mucosal symptoms was negatively associated with concentration of MuA, total LPS, and shorter lengths of 3-hydroxy fatty acids from LPS, C14, C16, and C18. Onset of general symptoms was negatively associated with C18 LPS. Onset of school-related symptoms was negatively associated with C16 LPS, but positively associated with total fungal DNA. In general, bacterial compounds (LPS and MuA) seem to protect against the development of mucosal and general symptoms, but fungal exposure measured as fungal DNA could increase the incidence of school-related symptoms.

Fungal DNA, allergens, mycotoxins and associations with asthmatic symptoms among pupils in schools from Johor Bahru, Malaysia

While there is a large variation of prevalence of asthma symptoms worldwide, what we do know is that it is on the rise in developing countries. However, there are few studies on allergens, moulds and mycotoxin exposure in schools in tropical countries. The aims were to measure selected fungal DNA, furry pet allergens and mycotoxins in dust samples from schools in Malaysia and to study associations with pupils' respiratory health effects. Eight secondary schools and 32 classrooms in Johor Bahru, Malaysia were randomly selected. A questionnaire with standardized questions was used for health assessment in 15 randomly selected pupils from each class. The school buildings were inspected and both indoor and outdoor climate were measured. Dust samples were collected by cotton swabs and Petri dishes for fungal DNA, mycotoxins and allergens analysis. The participation rate was 96% (462/480 invited pupils), with a mean age of 14 yr (range 14-16). The pupils mostly reported daytime breathlessness (41%), parental asthma or allergy (22%), pollen or pet allergy (21%) and doctor-diagnosed asthma (13%) but rarely reported night-time breathlessness (7%), asthma in the last 12 months (3%), medication for asthma (4%) or smoking (5%). The inspection showed that no school had any mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures. The mean building age was 16 yr (range 3-40) and the mean indoor and outdoor CO(2) levels were 492 ppm and 408 ppm, respectively. The mean values of indoor and outdoor temperature and relative humidity were the same, 29°C and 70% respectively. In cotton swab dust samples, the Geometric Mean (GM) value for total fungal DNA and Aspergillus/Penicillium (Asp/Pen) DNA in swab samples (Cell Equivalents (CE)/m(2)) was 5.7*10(8) and 0.5*10(8), respectively. The arithmetic mean (CE/m(2)) for Aspergillus versicolor DNA was 8780, Stachybotrys chartarum DNA was 26 and Streptomyces DNA was 893. The arithmetic means (pg/m(2)) for the mycotoxins sterigmatocystin and verrucarol were 2547 and 17, respectively. In Petri dish dust samples, the GM value for total fungal DNA and Asp/Pen DNA (CE/m(2) per day) was 9.2*10(6) and 1.6*10(6), respectively. The arithmetic mean (CE/m(2) per day) for A. versicolor DNA was 1478, S. chartarum DNA was 105 and Streptomyces DNA was 1271, respectively. The GM value for cat (Fel d1) allergen was 5.9 ng/m(2) per day. There were positive associations between A. versicolor DNA, wheeze and daytime breathlessness and between Streptomyces DNA and doctor-diagnosed asthma. However, the associations were inverse between S. chartarum DNA and daytime breathlessness and between verrucarol and daytime breathlessness. In conclusion, fungal DNA and cat allergen contamination were common in schools from Malaysia and there was a high prevalence of respiratory symptoms among pupils. Moreover, there were associations between levels of some fungal DNA and reported respiratory health in the pupils.

Fungal DNA in hotel rooms in Europe and Asia—associations with latitude, precipitation, building data, room characteristics and hotel ranking

There is little information on the indoor environment in hotels. Analysis of fungal DNA by quantitative PCR (qPCR) is a new method which can detect general and specific sequences. Dust was collected through swab sampling of door frames in 69 hotel rooms in 20 countries in Europe and Asia (2007-2009). Five sequences were detected by qPCR: total fungal DNA, Aspergillus and Penicillium DNA (Asp/Pen DNA), Aspergillus versicolor (A. versicolor DNA), Stachybotrys chartarum (S. chartarum DNA) and Streptomyces spp. (Streptomyces DNA). Associations were analysed by multiple linear regression. Total fungal DNA (GM = 1.08 × 10(8) cell equivalents m(-2); GSD = 6.36) and Asp/Pen DNA (GM = 1.79 × 10(7) cell equivalents m(-2); GSD = 10.12) were detected in all rooms. A. versicolor DNA, S. chartarum DNA and Streptomyces DNA were detected in 84%, 28% and 47% of the samples. In total, 20% of the rooms had observed dampness/mould, and 30% had odour. Low latitude (range 1.5-64.2 degrees) was a predictor of Asp/Pen DNA. Seaside location, lack of mechanical ventilation, and dampness or mould were other predictors of total fungal DNA and Asp/Pen DNA. Hotel ranking (Trip Advisor) or self-rated quality of the interior of the hotel room was a predictor of total fungal DNA, A. versicolor DNA and Streptomyces DNA. Odour was a predictor of S. chartarum DNA. In conclusion, fungal DNA in swab samples from hotel rooms was related to latitude, seaside location, ventilation, visible dampness and indoor mould growth. Hotels in tropical areas may have 10-100 times higher levels of common moulds such as Aspergillus and Penicillium species, as compared to a temperate climate zone.