Article

Fungi in bottled water: A case study of a production plant

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

A one-year fungal survey of a water bottling plant was conducted in order to evaluate the incidence and fluctuations of the mycobiota. The dominant fungal genera in order of highest numbers isolated were Penicillium, Cladosporium and Trichoderma followed by Aspergillus, Paecilomyces, and others. As expected, the highest number of isolates were collected during the warmer months, particularly May and June. Indeed during these two months there were more fungi present in the water, indicating that during those times of the year when fungal contamination is high, 0.4 mm filters should be changed on a more regular basis. In order to assess whether contamination was single or multi-loci, molecular methods based on the PCR were used for Penicillium brevicompactum. Overall, fungal contamination arose from multiple sources. Some P. brevicompactum strains were very "alike" and were detected during different sampling times, indicating that they were endemic to the plant. There was no evidence to suggest that fungi detected in the source water passed through to other parts of the plant. However, there was evidence that fungal strains isolated from the water filter were detected elsewhere in the factory, confirming the need to change filters more regularly during periods of high fungal contamination. In order to improve quality control a HACCP programme was implemented and Best Practice Guidelines introduced.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... In recent years, owing to the intensification of water pollution and increasing attention paid to health problems, scholars have begun to study fungal pollution in water [23][24][25][26][27]. At present, research on fungal pollution in water bodies has mainly concentrated on bottled mineral water [28][29][30], tap water [26,31,32], and water supply systems [33,34]. ...
Article
Full-text available
Fungi pollution in water can lead to serious problems, such as turbidity, odor, food pollution, mycotoxin production, and increased opportunistic infections among people with an immune deficiency. Few studies have reported the fungi community composition, quantity of fungi, and origin of fungi in groundwater. To study the change of quantity and community composition of fungi in groundwater at different times of year, this study evaluated the number of fungi and dominant fungi genera in groundwater and the factors affecting fungi quantity. The results showed that 18 genera of fungi were observed in the study area’s groundwater, among which Penicillium (18–27%), Aspergillus (17–26%), Acremonium (12–28%) were the three most dominant. The numbers of dominant fungi genera were as follows: Penicillium (21–62 CFU/100 mL), Aspergillus (18–43 CFU/100 mL), and Acremonium (15–38 CFU/100 mL). The number of fungi in water closely correlates with environmental variables such as pH, dissolved oxygen (DO), turbidity, and total organic carbon (TOC). Various genera of fungi were affected differently by unique environmental variables. The fungi in the water were also affected by components of the external environment, such as rainfall, surface farming, surface water sources, and so on. This study aims to provide meaningful information for understanding fungi pollution in groundwater.
... Certain fungi, e.g., Aspergillus and Candida, pose serious health concerns for hospitals and health institutions, particularly immunocompromised patients [107,108]. Presence of fungi in DWDS and BW have been reported previously [109][110][111]. ...
Article
Full-text available
The microbiological content of drinking water traditionally is determined by employing culture-dependent methods that are unable to detect all microorganisms, especially those that are not culturable. High-throughput sequencing now makes it possible to determine the microbiome of drinking water. Thus, the natural microbiota of water and water distribution systems can now be determined more accurately and analyzed in significantly greater detail, providing comprehensive understanding of the microbial community of drinking water applicable to public health. In this study, shotgun metagenomic analysis was performed to determine the microbiological content of drinking water and to provide a preliminary assessment of tap, drinking fountain, sparkling natural mineral, and non-mineral bottled water. Predominant bacterial species detected were members of the phyla Actinobacteria and Proteobacteria, notably the genera Alishewanella, Salmonella, and Propionibacterium in non-carbonated non-mineral bottled water, Methyloversatilis and Methylibium in sparkling natural mineral water, and Mycobacterium and Afipia in tap and drinking fountain water. Fecal indicator bacteria, i.e., Escherichia coli or enterococci, were not detected in any samples examined in this study. Bacteriophages and DNA encoding a few virulence-associated factors were detected but determined to be present only at low abundance. Antibiotic resistance markers were detected only at abundance values below our threshold of confidence. DNA of opportunistic plant and animal pathogens was identified in some samples and these included bacteria (Mycobacterium spp.), protozoa (Acanthamoeba mauritaniensis and Acanthamoeba palestinensis), and fungi (Melampsora pinitorqua and Chryosporium queenslandicum). Archaeal DNA (Candidatus Nitrosoarchaeum) was detected only in sparkling natural mineral water. This preliminary study reports the complete microbiome (bacteria, viruses, fungi, and protists) of selected types of drinking water employing whole-genome high-throughput sequencing and bioinformatics. Investigation into activity and function of the organisms detected is in progress.
... Ribeiro and colleagues conducted a 12-month survey of a drinking water bottling plant in Portugal to evaluate the diversity of the mycobiota [25]. The predominant fungal genera ranked in order of the highest numbers isolated were Penicillium, Cladosporium, and Trichoderma, followed by Aspergillus, Paecilomyces, and others. ...
Article
Full-text available
Aspergillus conida are ubiquitous in the environment, including freshwater, water for bathing, and in drinking water. Vulnerable patients and those suffering from allergic diseases are susceptible to aspergillosis. Avoidance of Aspergillus is of paramount importance. Potential outbreaks of aspergillosis in hospital facilities have been described where the water supply has been implicated. Little is known regarding the risk of exposure to Aspergillus in water. How does Aspergillus survive in water? This review explores the biofilm state of Aspergillus growth based on recent literature and suggests that biofilms are responsible for the persistence of Aspergillus in domestic and healthcare facilities’ water supplies.
... However, immunodeficient patients are widely susceptive to fungal infections and some previous works have reported the isolation of fungi in bottled water (7,19,26,30). ...
Article
Full-text available
The quality of mineral water commercialized in Brazil regarding the microbial content was analyzed and the results were compared with the standards established by the current legislation. Results demonstrated there was no bacterial contamination, but several types of fungi were found. Therefore, bottled mineral water could be considered a possible route for the transmission of filamentous fungi and yeasts.
... Fungal contamination of DW has been reported for decades, although investigations have been inadequate compared to those performed with bacteria (Paterson & Lima 2005;Hageskal et al. 2009). Some of the problems associated with fungal growth in DWDS are unsightly appearance, blocked pipes, odours and tastes, pigments, a source of potentially pathogenic and allergy-causing fungi, and mycotoxin production (Paterson & Lima 2005Ribeiro et al. 2006;Hageskal et al. 2009). However, the health problems associated with the presence of fungi in DWDS are still underestimated and not fully understood (Doggett 2000;Paterson & Lima 2005; Gonçalves et al. 2006;Hageskal et al. 2009). ...
Article
Full-text available
Current knowledge on drinking water (DW) biofilms has been obtained mainly from studies on bacterial biofilms. Very few reports on filamentous fungi (ff) biofilms are available, although they can contribute to the reduction in DW quality. This study aimed to assess the dynamics of biofilm formation by Penicillium expansum using microtiter plates under static conditions, mimicking water flow behaviour in stagnant regions of drinking water distribution systems. Biofilms were analysed in terms of biomass (crystal violet staining), metabolic activity (resazurin, fluorescein diacetate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide [MTT]) and morphology (epifluorescence [calcofluor white M2R, FUN-1, FDA and acridine orange] and bright-field microscopies). Biofilm development over time showed the typical sigmoidal curve with noticeable different phases in biofilm formation (induction, exponential, stationary, and sloughing off). The methods used to assess metabolic activity provided similar results. The microscope analysis allowed identification of the involvement of conidia in initial adhesion (4 h), germlings (8 h), initial monolayers (12 h), a monolayer of intertwined hyphae (24 h), mycelial development, hyphal layering and bundling, and development of the mature biofilms (≥48 h). P. expansum grows as a complex, multicellular biofilm in 48 h. The metabolic activity and biomass of the fungal biofilms were shown to increase over time and a correlation between metabolism, biofilm mass and hyphal development was found.
... Fungi have also been reported from bottled drinking water. 211,212 In the last decade, several more studies have resulted in increased knowledge on the occurrence of fungi in drinking water. 213−223 Statistically, it has been established that the odds for fungal recovery are three times higher in surfaces-sourced water compared with ground-sourced water, and fungi are commonly recovered from cold water and shower water than from hot tap water. ...
Chapter
There are over 100 different types of pathogens that can be found in contaminated water. Contaminated drinking water due to inadequate and unsanitary disposal of sewage and excreta continue to pose a threat to the health in many communities in developing countries. Groundwaters, surface waters, and distribution systems are at risk. Waterborne disease outbreaks are rising due to increasing vulnerable populations, political upheaval, and high numbers of refugees in developing countries. Natural disasters such as flooding and droughts due to climatic changes may also be affecting global water quality. As we move into the next century, it will be important to arm drinking water utility personnel with current and comprehensive information regarding waterborne pathogens and the importance of maintaining vigilance in their control.
... The abundance of Trichoderma species is also known in natural and artificial sweet water environments, e.g. in an acid mine drainage lake in Anhui Province, China or in bottled water (Ribeiro et al., 2006). In a study about the diversity and significance of mold species in Norwegian drinking water, besides Penicillium and Aspergillus, Trichoderma was also among the fungi with the highest maximum number of CFU in drinking water samples (Hageskal et al., 2006). ...
Chapter
This chapter attempts to summarize the recently available and rapidly increasing amount of information in the literature about the occurrence and biodiversity of Trichoderma species in different ecological habitats. Members of the genus are common in soil and rhizosphere of plants in natural and agricultural fields and forests and on decaying wood. They are also occurring in the air, settled dust and different water-related habitats including marine environments and drinking water. Furthermore, certain species are known as endophytes of plants, colonizers of mushroom-related natural and artificial substrata and facultative pathogens of humans, demonstrating a high adaptability to various ecological niches.
... This study exemplifies how microenvironments in industrial facilities have the potential to harbor recalcitrant microorganisms and initiate recurrent contamination. Although stringent management of classic transmission routes such as HVAC systems, municipal water supplies, and personnel traffic [7,13,15,17] are essential for ensuring process fidelity, acute awareness of microbial ecology, particularly the ability of diverse microorganisms to grow as biofilms, is needed to ensure all potential sources of contamination are identified and mitigated. Facilities maintenance programs can be undermined by the presence of microenvironments that serve as havens for contaminant strains, particularly those that are adept at biofilm formation. ...
Article
Fungal contamination of biomedical processes and facilities can result in major revenue loss and product delay. A biomedical research facility (BRF) culturing human cell lines experienced recurring fungal contamination of clean room incubators over a 3-year period. In 2010, as part of the plan to mitigate contamination, 20 fungal specimens were isolated by air and swab samples at various locations within the BRF. Aspergillus niger and Aspergillus fumigatus were isolated from several clean-room incubators. A. niger and A. fumigatus were identified using sequence comparison of the 18S rRNA gene. To determine whether the contaminant strains isolated in 2010 were the same as or different from strains isolated between 2007 and 2009, a novel forensic approach to random amplified polymorphic DNA (RAPD) PCR was used. The phylogenetic relationship among isolates showed two main genotypic clusters, and indicated the continual presence of the same A. fumigatus strain in the clean room since 2007. Biofilms can serve as chronic sources of contamination; visual inspection of plugs within the incubators revealed fungal biofilms. Moreover, confocal microscopy imaging of flow cell-grown biofilms demonstrated that the strains isolated from the incubators formed dense biofilms relative to other environmental isolates from the BRF. Lastly, the efficacies of various disinfectants employed at the BRF were examined for their ability to prevent spore germination. Overall, the investigation found that the use of rubber plugs around thermometers in the tissue culture incubators provided a microenvironment where A. fumigatus could survive regular surface disinfection. A general lesson from this case study is that the presence of microenvironments harboring contaminants can undermine decontamination procedures and serve as a source of recurrent contamination.
... Fungal strains isolated from commercial mineral water belong to various genera, such as Penicillium, Cladosporium, Fusarium, Acremonium, Alternaria, and Paecilomyces (1,2,7), and fungi contaminated 1% of commercial bottled mineral water at concentrations of more than 10 2 CFU/ml (7). A survey of a water bottling plant reported that Penicillium, Cladosporium, Aspergillus, Paecilomyces, Eurotium, and Talaromyces contaminated multiple sources, including source water in the plant (17). It was also reported that visible colonies grew from mold spores of Penicillium, Cladosporium, and Alternaria inoculated in commercial bottled mineral water under storage condition for a few months (3,6) and that strains of Alternaria alternata and Penicillium citrinum isolated from bottled mineral water produced mycotoxins in vitro (3). ...
Article
Full-text available
In recent years, bottled mineral water has undergone inactivation by methods other than the traditional heat treatment during the production process; there are fewer reports of the effectiveness of these inactivation methods on yeasts and molds in mineral water than on bacteria and protozoan oocysts. In this study, we evaluated the effects of UV irradiation and ozone treatment compared with heat treatment at 85 degrees C on yeast cells and mold spores inoculated into mineral water. A 5-log reduction occurred at a UV radiation dose of 31,433 microJ/cm2 for Saccharomyces cerevisiae and at 588,285 microJ/cm2 for Penicillium pinophilum. The treatment time for 5-log reduction estimated for UV irradiation was about 0.6 min for S. cerevisiae and about 10.7 min for P. pinophilum; at an ozone concentration of 0.1 ppm, it was 1.75 min for S. cerevisiae and 2.70 min for P. pinophilum, and at a concentration of 0.6 ppm, it was 0.32 min for S. cerevisiae and 0.57 min for P. pinophilum. Comparison of the inactivation effects among the three methods showed that UV irradiation and ozone treatment were less effective than heat treatment at 85 degrees C. Thus, when UV irradiation and ozone treatment are used for inactivation of mineral water, it seems that they need to be combined with heat treatment to achieve a definite effect. Yeast cells are more sensitive to all three inactivation methods than are mold spores, and the sensitivity of yeast cells and mold spores to these inactivation methods may vary among genera.
... Fungi have been reported from all types of water, from raw water to treated water, and from heavily polluted water to distilled or ultra-pure water. Fungi have also been reported from bottled drinking water (Cabral & Ferná ndez 2002; Fujikawa et al. 1997; Ribeiro et al. 2006). Statistically, it has been established that the odds for fungal recovery are three times higher in surfaces-sourced water compared with ground-sourced water, and fungi are more commonly recovered from cold water and shower water than from hot tap water (Hageskal et al. 2007). ...
Article
The occurrence of fungi in drinking water has received increased attention in the last decades, and fungi are now generally accepted as drinking water contaminants. The knowledge about the occurrence and diversity of fungi in water has increased considerably from a low knowledge base. However, the relevance of waterborne fungi for water quality and human health is poorly understood and still conflicting. Scientific reports on effective treatment against fungi in water are also few. This article presents a review of the literature on fungal water studies, including some general results, and considerations of significance, limits, contradictions, precautions, and practical consequences.
Article
Full-text available
Antifungal resistance remains a significant public health threat, yet poorly investigated. This study for the first time investigated the antifungal profile of moulds isolated from groundwaters in a rural community of Osun State, Southwest Nigeria. Groundwater samples were collected during dry and rainy seasons, and processed for mould isolation using standard methods. The isolates were presumptively identified and screened for in vitro susceptibility using 4 antifungals conventionally employed in clinical cases. Multiple antifungal resistance phenotypes and indices were equally evaluated. Representative of each putatively identified species were confirmed using molecular techniques. A total of 29 and 27 moulds were obtained during the dry and rainy seasons, respectively. Genus Aspergillus was the most prevalent (30.4%). Of the 56 isolates subjected to antifungal susceptibility testing, 55(98%) were resistant to fluconazole, followed by flucytosine 49(88%), ketoconazole 45(80%) and amphotericin B 31(55%). Exactly 46 isolates were multidrug-resistant, 23(50%) each, to 3 and 4 drugs, respectively. The multiple antifungal resistance index (MARI) evaluated was 1 in all the samples A-Q, except C (0.75), transcending the threshold limit of 0.2, indicating the isolates to be of high antifungal usage origin. The molecularly identified species had 99–100% similarity with reference strains from GenBank and evolutionary relationship of taxa revealed 10 distinct clades. The outcome of this study reveals a rise in the frequency of antifungal resistance in moulds towards standard drugs, thus, advocating for adequate investigations targeted at monitoring antifungal resistance in groundwater milieus.
Article
Due to the drift towards modern life style and poor quality of available supply water there has been a tremendous increase in the use of packaged drinking water in the recent decades. Packaged drinking water comes in various shapes and form which include plastic bottles, glass water, large gallons, jars and sachets. Before these are packed, the water pass through a series of purification processes as per the standards set by the national and international bodies. But recent studies have shown that the microbiological quality of even the packaged water cannot be trusted blindly. Hazardous microbial contamination has been found during earlier studies. This study was conducted to assess the microbiological quality of the different packaged water available throughout the markets of Allahabad city which included a total of 20 samples belonging to 5 national and international brands. The study showed that heavy number of coliform (E. coli), Staphylococcus aureus and faecal contamination (Streptococci) was found in the packaged drinking water and hence this water is not completely fit for human consumption. Consumption of such water may lead to several health hazards and serious diseases. This calls for an urgent need of quality assessment and monitoring of these packaged water by the local and national government bodies during their manufacturing and distribution.
Research
Full-text available
Background and purpose: One of the most prominent concerns for the water consumers is pathogenic microorganism contamination. Wells and underground water resources are the main resources of drinking water in Sari city, Iran. The main objectives of the research project were to explore the distribution and frequency of mycoflora in wells and underground water resources of the city and their contamination effects on humans. Materials and methods: Three reservoirs and 18 wells or underground water resources were analyzed. Water samples were then filtered and analyzed according to the World Health Organization guidelines. Each filter and 0.2 ml of suspension inoculated on SDA+CG media. For fungal growth, plates were incubated at 27’C for 7-10 days. The fungi were identified by standard mycological techniques. Results: Fungal colonies were isolated from all samples. From total of 160 fungal colonies isolated from wells water, 14 species of fungi were distinguished. Rhodotorula (54.4%), Monilinia (13.7%), Alternaria (6.9%) were the most commonly isolated. Drechslera, Rhizopus, and Exserohilum (0.6%) had the lowest frequency. There was no significant difference between fungal elements isolated from three major reservoirs (P>0.05). Conclusion: This study revealed that resources of drinking water from an area have to monitored and if its fungal CFU be greater than a certain value, medical and health preventive measures should be taken before the water is used by human. In this context, public and private awareness should also be provided through the media, broadcasting, teachers and scholars.
Article
The presence of molds in the winery is of concern for winemakers, as molds can be responsible for ochratoxin A and cork taint in wines. The presence of airborne molds was analyzed in four different areas (cask, vinification, bottle cellar, and bottling line) in three Rioja appellation wineries with different characteristics of design, output capacity, and age. Samples were taken four times during the year (winter, spring, summer, and fall). Winery design was a determinant factor in the levels of relative humidity and temperature, which determined the total counts of airborne molds. However, design did not influence the distribution of the main types of molds (Penicillium and Cladosporium) found in the different areas studied. The activity conducted in each part of the winery, mainly when grapes were delivered and when auxiliary materials were handled, affected the total number of airborne molds present at specific times. Hence, a relationship could be established between the activity in the winery and the diversity index. The diversity index was much higher in the older wineries than in the most recently constructed winery. Results indicate the variables that need to be considered in order to control levels of airborne molds in wineries, including the design needed to allow the establishment of an effective system for controlling temperature and relative humidity. © 2014 by the American Society for Enology and Viticulture. All rights reserved.
Article
The microbiological quality of bottled water has been studied over the past 30 years as bottled water can contain pathogens. The aim of this study was to determine if retailed bottled water in New Zealand complied with the Australia and New Zealand Food Standards (ANZFS) Code (2002) and the New Zealand Microbiological Reference Criteria (1995). Thirty-eight domestic and imported bottled water brands were tested for Total Coliforms, Escherichia coli, Pseudomonas aeruginosa, Enterococci, HPC, Yeasts and Moulds. Three brands did not comply with both of the above criteria for Total Coliforms. Seventeen brands did not comply with HPC criteria and 21 brands displayed mould growth. A questionnaire was used to assess the impact of manufacturing procedures on bottled water quality. Four responding manufacturers, which represented 11 bottled water brands, bottled at least one brand that did not comply with the ANZFS Code for HPC. Our study demonstrated that even 10 years after the initial study in New Zealand microbiological contamination in bottled waters was still being detected. We further demonstrated that monitoring of bottled water microbiological quality was essential and that the presence of manufacturers' procedures for ensuring satisfactory bottled water microbiological quality did not always guarantee it.
Article
Full-text available
The quality of mineral water commercialized in Brazil regarding the microbial content was analyzed and the results were compared with the standards established by the current legislation. Results demonstrated there was no bacterial contamination, but several types of fungi were found. Therefore, bottled mineral water could be considered a possible route for the transmission of filamentous fungi and yeasts.
Article
Unlabelled: This paper studies the presence of mold in the air of a vinification and ageing wine cellar. The influence of other factors such as the time of year, the sampling point, and the activity being carried out in the cellar has been analyzed. Neither the type of activity being carried out in the cellar nor the temperature or relative humidity fluctuations throughout the year are determining factors in the presence of mold in the air. For this group of microorganisms, the design of the cellar studied is the fundamental factor. Areas with little ventilation favor high levels of relative humidity and, hence, a higher presence of mold in the air. The mold population in these areas is not very diverse, which indicates that colonization by certain types of mold that have adapted to the conditions established therein is permanent. Areas with greater air flow, constant activity, and frequent cleaning show lower mold populations in the air and of a more varied composition. Practical application: This work shows that given the growing importance of the presence of mold in wine cellars, the design thereof should take into account suitable ventilation of all the areas and control of the relative humidity. Hence, the presence of traditional underground areas for ageing wine, which is justifiable in seasons where temperature and humidity control lead to major technical problems, should be reconsidered in the design of new wine cellars.
Article
We report the repeated isolation for Trichoderma.harzianum, a rare opportunistic pathogen from three sets of each of the following clinical samples; blood serum, skin lesions, sputum and throat of a pediatric ALL patient with neutropenia. The definition of invasive fungal infection requires evidence of the presence of fungal elements in tissue samples, in addition to the isolation of suspected etiologic agent in culture. However, invasive procedures are not always applicable due to several factors, as for example in our case, the poor general status of the individual patient or thrombocytopenia. The present paper also emphasizes the problems encountered in obtaining appropriate samples and diagnosing invasive fungal disease in immunocompromised patient populations, including those with hematological malignancy. Three cases involving T. harzianum, including this one, have been described thus far in the literature. All were fatal and the fungus was resistant to antifungal therapy. A critical review of the other two cases of Trichoderma infections in humans is provided.
Article
Full-text available
This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected only-fungal sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.
Article
The genetic relatedness among 41 isolates of Rhizoctonia solani belonging to 11 AGs was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by 3 RAPD primers (P14, R28 and RC09), ERIC (ERICIR-I/ERIC2) and REP (REP1R-I/REP2-I) gene sequences. Based on the banding patterns of PCR-amplified products, seven putative groups among the 41 isolates were recognized. RAPD-PCR generated multiple distinct products showing considerable variability among the isolates of different AG types. Isolates originated from the same geographical origin or host plants were not always genetically related. Amplification with ERIC and REP elements enabled detection of AGs/subgroup-conserved and isolate-polymorphic variants, respectively. Though pattern types generated by ERIC and REP in general, were distinctly variable among different subgroups, less variable fingerprint patterns were produced within the isolates of each subgroup.
Article
Molecular approaches for the assessment of intraspecific diversity within an economically important plant pathogen were compared with traditional physiological methods (vegetative compatibility testing). The vegetative compatibility groups (VCGs) of 14 isolates of Fusarium oxysporum f.sp. cubense (FOC) from Kenya were first assessed using nitrate non-utilizing mutants. Nine of these isolates, from different areas of the country, were compatible with one or more of VCGs 0124, 0125, 0128 and 01220, i.e. they formed a single clonal lineage. Three isolates, all originating from the banana growing district of Kisii, were compatible with the VCG 01212 and formed a second distinct clonal lineage. Mutants could not be recovered from one isolate (62) and two isolates (27 and 30) were not vegetatively compatible with any of the VCG testers and may represent two novel VCGs. Polymerase chain reaction (PCR) fingerprinting, especially when using the M13 derived primer, was found to produce banding patterns that correlated with clonal lineage and also distinguished isolates 27 and 30 when analysed by unweighted pair group method analysis and principle co-ordinate analysis. This approach also distinguished FOC from F. oxysporum IMI350438 isolated from Triticum sp. and from isolates of Colletotrichum gloeosporioides. Total protein profiles were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and although clonal lineages were not separated, isolates 27 and 30 were again distinguishable and FOC produced a different profile to F. oxysporum (IMI 350438) and C. gloeosporioides.
Article
One M13 phage derived and three simple sequence repeat primers were assessed for the development of a standard set of PCR primers for population studies with filamentous fungi. Fungal isolates from five plant or insect pathogenic genera with ascomycete and basidiomycete affinities were screened. Three of the four primers were found to be suitable for generating ‘genetic fingerprints’ for all of the fungal genera tested. Strain groupings obtained from the individual primers were found to be supportive through constrained two-dimensional cluster analysis.
Chapter
Fungi are a ubiquitous and diverse group of unique organisms accepted as a fifth kingdom. They are associated intrinsically with water, and so it is not surprising that they can occasionally cause problems. Two areas exist: (a) natural contamination and (b) unnatural contamination. Natural contamination is the equivalent of that which occurs with bacteria. Unnatural, or intentional, contamination is associated predominately with security in a field that has rapidly ascended the agenda. Furthermore, bottled water is a huge and growing commercial market and is susceptible to fungal contamination. A major problem is visual growth, which is what is presented to the consumer. Whereas this may not be apparent to the consumer of tap water, it is obvious in bottled water. However, there are technical difficulties associated with quantification and identification. Other actual issues can be odor and pigmentation. In addition, certain fungi cause allergic reactions and some are animal pathogens, including human pathogens, which are of particular concern to immunocompromised people. Finally, toxic compounds can be produced by fungi, which are referred to as mycotoxins. Aflatoxins have been demonstrated to occur in stored water naturally and other mycotoxins in in vitro experimentation. Obviously, it is mycotoxin contamination that is the biggest security threat. Methods are provided in this entry for the isolation and biochemical analysis of fungi and the properties of significance to drinking water. The use of a fungal lipid called ergosterol to quantify fungi is preferred. In conclusion, fungi in drinking water are often ignored but require a great deal more attention, particularly in relation to security threats.
Article
SummaryA novel method, Random Amplified Microsatellites (RAMS, due to the nature of amplified markers as two randomly amplified microsatellites with the intervening sequence), was applied to generate DNA markers in a variety of fungi (Armillaria cepistipes, Gremmeniella abietina, Heterobasidion annosum, Pbytophthora cactorum, Phlebiopsis gigantea, and Stereum sanguinolentum). It is based on the polymerase chain reaction (PCR), and uses primers containing microsatellite sequences and degenerate anchors at the 5' end. The method is highly reproducible, applicable to all tested fungal species including members of the Phycomycetes, Ascomycetes and Basidiomycetes, and allows detection of interspecific and intraspecific DNA-polymorphisms.
Article
To breed or select banana cultivars with durable resistance to Fusarium wilt it is necessary to determine the genetic variation that exists within the pathogen, Fusarium oxysporum f. sp. cubense, as potentially resistant cultivars ideally should have resistance to all variants of the pathogen. We have analysed genetic variation within a world-wide collection of isolates of F. oxysporum f. sp. cubense which represented races 1, 2 and 4 and 11 different vegetative compatibility groups (VCGs) using RAPD-PCR. Comparison of the RAPD-PCR banding patterns both visually and by phenetic analysis sub-divided isolates of F. oxysporum f. sp. cubense into two major groups. Group 1 contained isolates belonging in VCGs 0120, 0121, 0122, 0126, 01210, 01211 and 01212 and group 2 contained isolates belonging in VCGs 0123, 0124, 0124/5 and 0125. The RAPD-PCR banding patterns were generally VCG specific and it was possible to determine the genetic relatedness between the different VCGs and of isolates within each VCG. The two groups differentiated by RAPD-PCR analysis correspond with previous classifications based on volatile production and electrophoretic karyotyping. In contrast, there was no correlation between RAPD-PCR banding pattern and race. The results support the hypothesis that F. oxysporum f. sp. cubense co-evolved with edible bananas and their wild diploid progenitors in South East Asia.
Article
Thirty-eight strains of the entomopathogenic Beauveria bassiana, isolated from diverse species of Lepidoptera (Pyralidae) or Coleoptera (Curculionidae, Chrysomelidae, and Scolytidae) from various geographical sites, were examined by RFLP and RAPD analyses. Similar groupings were recovered from both approaches and these showed clear relationships between the population structure of B. bassiana and some defined host species. Strains isolated from members of the Pyralidae were recovered as two main groups, one group consisted of all strains isolated from Ostrinia irrespective of their origin. The second group consisted primarily of strains isolated from Diatraea in Cuba. All strains isolated from the curculionid genus Sitona clustered as a single distinct group, separated from strains from other curculionids. In contrast, strains isolated from the pyralid genus Maliarpha, and the coleopteran Chrysomelidae, gave heterogenous patterns and were not recovered as distinct groups. Groups from cluster analysis and non- hierarchic ordination methods were compared and the relative merits of the different grouping strategies are discussed.
Article
Suitability and reproducibility of ISSR-PCR to find strain-specific and taxon specific markers for strains of the tree-root endophytes Phialocephala fortinii and ’Type 1’, a non-sporulating dematiaceous mycelium, were examined. The results were compared with data previously generated by isozyme analyses. P. fortinii and ’Type 1’ are two DSE taxa and are abundant colonisers of coniferous forest-tree roots in the North Temperate zone. ’Type 1’ was never observed to sporulate in pure culture but is well defined by its cultural characteristics. DNA of 14 strains per taxon was amplified with three short, 17-18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) with two (CCA) or three degenerated bases at their 5’-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scored for absence/presence, respectively, and the binary matrix subjected to multiple correspondence analysis (MCA). ISSR-PCR was found to be highly reproducible since amplification of DNA from several single-hyphal-tip cultures of the same strain resulted in identical banding patterns. Eighty-five (92.4%) of the 92 DNA fragments were polymorphic. The fragments ranged from 320 to 4100 bp. ISSR-PCR was found to be a powerful tool to find strain-specific and taxon-specific markers. Each strain showed a unique banding pattern and diagnostic bands for the two taxa could be identified. ISSR-PCR data correlated neither with the geographical nor the host origin of the strains. The strains grouped into similar clusters independently of whether MCA was performed with ISSR-PCR or isozyme data. However, ISSR-PCR allowed the differentiation of strains with the same allozyme phenotype.
Article
Intra- and interspecific variation of thirty-two isolates assignable to the genus Beauveria was evaluated using 64 morphological and biochemical characters. Two isolates of Tolypocladium cylindrosporum were included to test generic concepts. Seventeen cluster groups were obtained following a numerical taxonomic analysis, each group being separated by at least one character. Cultural characters were highly variable and could not be used reliably for species determination. Spore form was the most useful criterion to distinguish between species. Biochemical data generally supported species concepts based purely on morphology, with the exception of B. bassiana which comprised a heterogeneous assemblage of strains. There is evidence from API ZYM and esterase patterns that this variability is determined by host (substrate) and geographical origins. B. alba, although morphologically close to B. bassiana, could be separated readily on biochemical characters using principal component analysis. The following species of Beauveria are recognized: B. alba, B. amorpha, B. bassiana, B. brongniartii, B. velata, B. vermiconia and an undescribed taxon close to B. amorpha but morphologically and biochemically distinct. Both isolates of T. cylindrosporum cluster in the same group and, on the basis of present evidence, particularly conidiogenesis, the synonymy of this genus with Beauveria is questioned.
Article
The densities of filamentous fungal colonies, together with physicochemical and bacteriological parameters, were assessed in a chlorinated and unchlorinated drinking water distribution system at eight separate times over a period of 1 year. Filamentous fungal colonies were enumerated by membrane filtration on Czapek-Dox agar. The mean number of filamentous fungal colony-forming units per 100 mL of drinking water was 18 in the unchlorinated and 34 in the chlorinated system. The majority of filamentous fungi isolated wee saprophytic Deuteromycotina. The four most frequently occurring genera were Penicillium, Sporocybe, Acremonium, and Paecilomyces. In the chlorinated system, only physicochemical parameters correlated with observed fungal frequencies, whereas in the unchlorinated system, none of the parameters exhibited significant correlations with fungal numbers.
Article
The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.
Article
Evolutionary conservation of an interspersed repetitive DNA sequence, BOX, from Streptococcus pneumoniae was investigated to explore the mosaic nature of these elements. BOX elements consist of various combinations of three subunits, boxA, boxB, and boxC. Eight oligonucleotide probes were designed based on consensus DNA sequences of boxA, boxB, and boxC subunits. DNA hybridization studies and PCR using these probes/primers demonstrate that oligonucleotide sequences within the boxA subunit appear to be conserved among diverse bacterial species. The boxB and boxC subunits show only limited, if any, sequence conservation in bacteria other than S. pneumoniae. Intact BOX elements with boxA, boxB, and boxC subunits were only present in high copy number in pneumococcal strains. This pattern of differential conservation lends support to the modular nature of BOX repetitive elements in that boxA-like subsequences are effectively independent of boxB-like or boxC-like subunits in bacteria other than S. pneumoniae. Furthermore, dendrograms derived from repetitive sequence-based PCR (rep-PCR) fingerprints of S. pneumoniae isolates using the BOXA1R primer yielded clustering patterns that were similar to those obtained previously by other methods, suggesting that these repetitive sequence-based DNA fingerprints represent intrinsic properties of an S. pneumoniae strain's genome. Our results indicate widespread conservation of boxA-like subsequences in the bacterial kingdom, lend support to the mosaic nature of BOX in S. pneumoniae, and demonstrate the utility of boxA-based primers for rep-PCR fingerprinting of many microorganisms.
The microbiology of water Part 1: Drinking water, Report on Public Health and Medical Subjects No.71, Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office
  • Anonymous
Anonymous. The microbiology of water. Part 1: Drinking water, Report on Public Health and Medical Subjects No.71, Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office, 1994. References
Report DW-01/F for UK Water Industry Research Limited. Egham, International Mycological Institute
  • J Kelley
  • G Hall
  • Rrm Paterson
Methods for the examination of waters and associated materials. London, Her Majesty's Stationery Office
  • Anonymous