Mycoflora isolation and molecular characterization of Aspergillus and Fusarium species in Tunisian cereals

Wheat, barley and maize are the mainly consumed cereals in Tunisia. This study aimed to determine the mycoflora of these cereals with special focus on the mycotoxigenic Aspergillus and Fusarium species. Freshly harvested samples and other stored samples of each type of cereal (31 and 34 samples, respectively) were collected in Tunisia and cultured for fungal isolation and identification. Identification of fungal genera was based on morphological features. Aspergillus and Fusarium species were identified by species specific PCR assays complemented with DNA sequencing. Alternaria (70.83%), Eurotium (62.50%), Aspergillus (54.17%) and Penicillium (41.67%) were the most frequent fungi isolated from wheat. Penicillium (75%), Aspergillus (70%), Eurotium (65%) and Alternaria (65%) were the most frequently recovered genera from barley. The predominant genera in maize were Aspergillus (76.19%), Eurotium (42.86%), and Penicillium (38.09%). Aspergilllus, Penicillium, Fusarium and Alternaria were detected in both stored and freshly harvested grain samples. The frequencies of contamination with Aspergillus, Fusarium and Alternaria were higher in freshly harvested samples, whereas Penicillium species were more frequent in stored samples. The predominant Aspergillus species detected were A. flavus and A. niger. The Fusarium species detected were F. equiseti, F. verticillioides, F. nygamai, and F. oxysporum. This study suggested the potential risk for Aflatoxins and, to a lesser extent, for Ochratoxin A in Tunisian cereals. This is the first survey about mycoflora associated with wheat, barley and maize in Tunisia.     

Enumeration of airborne molds in some indoor environments of Madras city (India) by cultural and non-cultural volumetric samplers

The air mycoflora of six indoor environments in Madras city (India) has been investigated by sampling air with an Andersen sampler and a Burkard personal sampler. Forty-eight species assignable to 24 genera were recorded by Andersen sampler. Spores belonging to 14 genera in addition toPenicillium andAspergillus were identified from Burkard trap slides. Species ofAspergillus, Penicillium, Mucor andRhizopus were most frequently isolated in considerable numbers. As a single genusAspergillus ranked first followed byPenicillium at some sites, andCladosporium at some other sites. The predominance ofPenicillium andAspergillus was also confirmed by Burkard trap data. Spores belonging toGanoderma, Nigrospora, Epicoccum, andTetraploa were recorded only by Burkard sampler, thereby suggesting the necessity of using two complimentary spore traps, cultural and non-cultural, in any aerobiological investigation.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Diversity and Drug Resistance of Filamentous Fungi Isolated from the Fresh Raspberries

Fungi are one of the most widely distributed microorganisms in the environment, including food such as fruits, vegetables and other crops, posing a potential threat to food safety and human health. The aim of this study was to determine the diversity, intensity and drug resistance of potentially pathogenic filamentous fungi isolated from the fresh raspberries (Rubus idaeus L.). A total of 50 strains belonging to genera Fusarium, Cladosporium, Alternaria, Penicillium, Mucor, Rhizopus, Aspergillus and Acremonium were tested for drug resistance against 11 antifungals by disc diffusion and gradient strips methods. The average mycological contamination in the examined samples of raspberries amounted to 4.34 log CFU/g. The Cladosporium was isolated from all tested samples, followed by Alternaria and Fusarium with a frequency of 61% and 34%, respectively. The highest level of drug resistance was observed for Acremonium genera and Fusarium strains recorded a wide variation in drug resistance as revealed by susceptibility with amphotericin B and voriconzole with MICs ranged from 0.5–4 µg/ml and posaconazole with MICs ranging from 3–8 µg/ml. All fungal strains showed 100% resistance to caspofungin, fluconazole and flucytosine with both the methods, and 100% resistance to micafungin and anidulafungin in the gradient strip method.     

Predicting disease occurrence with high accuracy based on soil macroecological patterns of Fusarium wilt

Soil-borne plant diseases are increasingly causing devastating losses in agricultural production. The development of a more refined model for disease prediction can aid in reducing crop losses through the use of preventative control measures or soil fallowing for a planting season. The emergence of high-throughput DNA sequencing technology has provided unprecedented insight into the microbial composition of diseased versus healthy soils. However, a single independent case study rarely yields a general conclusion predictive of the disease in a particular soil. Here, we attempt to account for the differences among various studies and plant varieties using a machine-learning approach based on 24 independent bacterial data sets comprising 758 samples and 22 independent fungal data sets comprising 279 samples of healthy or Fusarium wilt-diseased soils from eight different countries. We found that soil bacterial and fungal communities were both clearly separated between diseased and healthy soil samples that originated from six crops across nine countries or regions. Alpha diversity was consistently greater in the fungal community of healthy soils. While diseased soil microbiomes harbored higher abundances of Xanthomonadaceae, Bacillaceae, Gibberella, and Fusarium oxysporum, the healthy soil microbiome contained more Streptomyces Mirabilis, Bradyrhizobiaceae, Comamonadaceae, Mortierella, and nonpathogenic fungi of Fusarium. Furthermore, a random forest method identified 45 bacterial OTUs and 40 fungal OTUs that categorized the health status of the soil with an accuracy >80%. We conclude that these models can be applied to predict the potential for occurrence of F. oxysporum wilt by revealing key biological indicators and features common to the wilt-diseased soil microbiome.Subject terms: Molecular ecology, Infectious-disease diagnostics     

Effects of concrete properties and nutrients on fungal colonization and fouling

This study describes the fouling of concrete surfaces by diverse fungal genera under controlled laboratory conditions. A circulating flow-through chamber was designed for testing the effects of different concrete compositions and exogenously added nutrients on fungal colonization and fouling. Fungal strains belonging to the genera Alternaria, Cladosporium, Epicoccum, Fusarium, Mucor, Penicillium, Pestalotiopsis, and Trichoderma were cultured directly from visibly fouled concrete structures and used individually and in a mix to inoculate mortar tiles varying in cement composition, supplementary cementitious material additions, water-to-cement ratio, and surface roughness. A strong positive relationship was observed between tile water-to-cement ratio and the amount of biofouling. In addition, cement containing photocatalytic titanium dioxide and exposed to artificial sunlight strongly inhibited fungal colonization and fouling. Mortar tiles coated with form-release oil and incubated with sterile rainwater were also capable of supporting fungal colonization. Our results indicate that the fouling of concrete surfaces by fungi can be influenced by variations in concrete composition variations and available nutrients.     

The biogeography of fungal communities in wetland sediments along the Changjiang River and other sites in China

Whether fungal community structure depends more on historical factors or on contemporary factors is controversial. This study used culture-dependent and -independent (polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)) methods to assess the influence of historical and contemporary factors on the distributions of fungi in the wetland sediments at 10 locations along the Changjiang River and at 10 other locations in China. The culture-dependent approach detected greater species diversity (177 operational taxonomic units (OTUs)) than PCR-DGGE analysis (145 OTUs), and the species in the genera of Penicillium (relative frequency=16.8%), Fusarium (15.4%), Aspergillus (7.6%), Trichoderma (5.8%) and Talaromyces (4.2%) were dominant. On the basis of DGGE data, fungal diversity along the Changjiang River increased from upstream to downstream; altitude explained 44.8% of this variation in diversity. And based on the data from all 20 locations, the fungal communities were geographically clustered into three groups: Southern China, Northern China and the Qinghai-Tibetan Plateau. Multivariate regression tree analysis for data from the 20 locations indicated that the fungal community was influenced primarily by location (which explained 61.8% of the variation at a large scale), followed by total potassium (9.4%) and total nitrogen (3.5%) at a local scale. These results are consistent with the concept that geographic distance is the dominant factor driving variation in fungal diversity at a regional scale (1000–4000 km), whereas environmental factors (total potassium and total nitrogen) explain variation in fungal diversity at a local scale (<1000 km).     

Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra

This multidisciplinary research combines knowledge in molecular biology with fungal morphology, aiming at the identification of infecting fungi from historical documents on the Archive of the University of Coimbra. The identification of infecting fungi on several bibliographic documents and support materials was based both on ribosomal DNA loci amplification and sequencing, and morphological identification, using macro- and microscopical traits. A high fungal diversity was found in all types of support: parchment, laid-paper and wood-pulp paper. Fourteen fungal genera were isolated, identified, and kept in culture. The most frequent were Cladosporium, Penicillium and Aspergillus, and other less frequent genera, such as Alternaria, Botrytis, Chaetomium, Chromelosporium, Epicoccum, Phlebiopsys and Toxicocladosporium were also present. Within these genera, 20 different species were identified, from which 15 were found only in a single support type. Cladosporium cladosporioides and Penicillium chrysogenum were the only species present in all support types.     

Fungi utilizing keratinous substrates

One of the basic tasks in the field of biodeterioration is to recognise the microbial species responsible for the destruction of particular substrates, and to identify factors impacting the level of damage caused by microorganisms. Even in 1839, it was known that there existed some fungi capable of attacking keratinized tissues, although, at that time, only dermatophytes were recognised. The relevant literature pertaining to microbiological deterioration of keratinous substrates includes 299 fungi belonging to 100 genera. Representatives of the genera Aspergillus, Penicillium, Chrysosporium, Fusarium, Microsporum, Trichophyton, and Acremonium appear to be the most common. Of the 299 species collected, 107 belonging primarily to the Onygenales and Eurotiales are pathogenic to humans. The research focusing on microbial ability to colonize and destroy keratinous materials has been carried out mainly on sheep fleece, hairs, and feathers, but only a few authors have dealt with woollen fabric biodeterioration, which is of particular significance for the preservation of antique textiles.     

Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers

Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.     

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins in the Indoor Environment

The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with known Stachybotrys chartarum contamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectable S. chartarum growth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal genera Aspergillus, Chaetomium, Cladosporium, Fusarium, Memnoniella, Penicillium, Rhizopus, and Trichoderma, five Stachybotrys strains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from <10 to >1,300 pg/m³ of sampled air. The control environments demonstrated statistically significantly (P < 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain of S. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist in Stachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.     

Microfungal Contaminants on Mobile Phones of Health Services Vocational School Students in Marmaris,Turkey

In this study, it was aimed to determine microfungi on mobile phones. Totally, 50 mobile phones were used belonging to Health Services Vocational School students. The samples were taken by swabbing the screen and keys of mobile phones using moistened sterile swab sticks. A total of 24 different microfungal species were obtained belonging to Alternaria, Aspergillus, Cladosporium, Geotrichum, Penicillium, Phoma, Rhinocladiella, Scopulariopsis, Trichoderma, and Trichophyton genera. The genera of microfungi most abundant in terms of the number of species on the mobile phones were Aspergillus, Cladosporium, and Penicillium. Numerically, Cladosporium was found as the most abundant on the mobile phones. Cladosporium herbarum colonies were highest in number, followed by Cladosporium sphaerospermum, and Penicillium verrucosum var. cyclopium. When percentages of each species present on the mobile phones were considered, C. herbarum and C. sphaerospermum were the most common. There was a great similarity between the dominant microfungi isolated from mobile phones and dominant microfungi obtained from studies of atmospheric microfungi in Turkey.     

Molecular characterization of airborne fungi in caves of the Mogao Grottoes, Dunhuang, China

In this study, we analyzed air samples collected from several sites within the Mogao Grottoes, Dunhuang, China. The samples were collected each month from September 2008 to August 2009 from an open cave (OC), a semi-open cave (SC), a closed cave (CC), and the entrance (EN) of the Mogao Grottoes. Sampling was carried out using a six-stage Andersen FA-I sampler; then samples were cultured and fungal isolates were identified by partial sequencing of their internal transcribed spacer (ITS) region. Eleven different fungal genera were found, and the most prevalent was Cladosporium, followed by Fusarium, Penicillium, Alternaria, and Aspergillus. The fungal community composition varied among the four sites. Fungal community structure was significantly related to site (r = −0.293, p = 0.039) and to time of year (r = −0.523, p = 0.000). The concentrations and abundance of airborne fungi varied greatly throughout the year at the four sampling sites. Meteorological parameters (e.g., temperature, relative humidity) and the number of visitors also influenced both abundance and community structure of airborne fungi in the Mogao Grottoes.     

Mycobiota and mycotoxins in fermented feed,wheat grains and corn grains in Southeastern Buenos Aires Province,Argentina

Wheat (as bran) and corn (as dry grain or fermented feed) are main ingredients of feedstuffs used in local cattle and pig farms in the South of the Buenos Aires Province (Argentina). Therefore, determining mycobiota and mycotoxins in wheat and corn is of prime importance for developing feed management techniques to optimise animal production and to minimize toxicity. Then, a mycological survey was carried out in the Southeastern part of the Buenos Aires Province, in order to identify the mycobiota and the main mycotoxins present in fermented feed, wheat grain and corn grain samples. Samples were cultured for fungal quantification, isolation and identification, and analysed for deoxynivalenol (DON), zearalenone (ZEA), T-2 toxin and aflatoxins (AFLA). Penicillium (74%), Aspergillus (32%) and Scopulariopsis (21%) were the prevalent genera in fermented feed. Penicillium (70%), Fusarium (47%) and Aspergillus (34%) were the most frequent fungi isolated from corn. Penicillium (42%), Fusarium (27%) and Alternaria (25%) were the most frequently recovered genera from wheat. DON was detected in 59% of the corn samples, in 45% of the wheat samples and in 38% of the silage samples. ZEA was detected in 36% of the corn samples, in 49% of the wheat samples and in 16% of the silage samples. T-2 toxin and aflatoxin B1 were each detected in 4% of the corn samples. Eighteen percent of the fermented feed samples showed T-2 contamination. Fermented feed and wheat samples were negative for AFLA.     

Mycoflora and mycotoxins of peanut (Arachis hypogaea L.) seeds in Egypt. III. Cellulose-decomposing and mycotoxin-producing fungi

From 40 peanut seed samples collected in Egypt, forty-three species and one variety of fungi, belonging to 16 genera, were collected. The most dominant genera were Aspergillus (11 species + one variety), Penicillium (11 species) and Fusarium (4 species). From the preceding genera A. fumigatus, A. flavus, A. niger, P. chrysogenum and F. oxysporum were the most frequent species.Forty-nine isolates belonging to 12 species and one variety were tested for production of mycotoxins, after growth on liquid medium containing two carbon sources (sucrose or cellulose). Thin layer chromatographic analysis revealed that the quality and quantity of mycotoxins was higher on sucrose than cellulose. Mycotoxins identified were aflatoxins B₁, B₂, G₁ & G₂, citrinin; fumagillin; diacetoxyscirpenol T-2 toxin; satratoxin H; and zearalenone.     

Contamination of common spices in Saudi Arabia markets with potential mycotoxin-producing fungi

Fifteen spices obtained from common markets were examined for their mould profile. A total of 520 fungal isolates, representing 57 species, were recovered and identified from dried and ground spice samples on three different media using standard dilution plate method. The most heavily contaminated spice samples examined were observed in ginger in order of magnitude of 5325–6800 cfu/g. The most predominant fungal genera encountered were Aspergillus, Penicillium, and Rhizopus. Relative occurrence values of taxa disclosed ranged between 80% for Aspergillusflavus, Aspergillusniger and Penicilliumarenicola, and 10% for some species. Samples obtained from sumac encountered very rare colony counts indicating its antifungal prosperities. The present magnitude of contamination and spectra of mycobiota approximate those reported for similar spice samples. Several potentially mycotoxigenic fungi were isolated from the majority of samples. The present study attracts the attention to potential risk for mycotoxins contamination may be caused as a result of using these spices, especially in great quantities. The study strongly recommends reduction in application of heavily contaminated spices like ginger in food processing and using some others like clove and sumac due to their antimicrobial properties.     

Identifying airborne fungi in Seoul,Korea using metagenomics

Fungal spores are widespread and common in the atmosphere. In this study, we use a metagenomic approach to study the fungal diversity in six total air samples collected from April to May 2012 in Seoul, Korea. This springtime period is important in Korea because of the peak in fungal spore concentration and Asian dust storms, although the year of this study (2012) was unique in that were no major Asian dust events. Clustering sequences for operational taxonomic unit (OTU) identification recovered 1,266 unique OTUs in the combined dataset, with between 223?96 OTUs present in individual samples. OTUs from three fungal phyla were identified. For Ascomycota, Davidiella (anamorph: Cladosporium) was the most common genus in all samples, often accounting for more than 50% of all sequences in a sample. Other common Ascomycota genera identified were Alternaria, Didymella, Khuskia, Geosmitha, Penicillium, and Aspergillus. While several Basidiomycota genera were observed, Chytridiomycota OTUs were only present in one sample. Consistency was observed within sampling days, but there was a large shift in species composition from Ascomycota dominant to Basidiomycota dominant in the middle of the sampling period. This marked change may have been caused by meteorological events. A potential set of 40 allergyinducing genera were identified, accounting for a large proportion of the diversity present (22.5?7.2%). Our study identifies high fungal diversity and potentially high levels of fungal allergens in springtime air of Korea, and provides a good baseline for future comparisons with Asian dust storms.     

Occurrence of Aspergillus section Flavi and aflatoxin B1 in corn genotypes and corn meal in Argentina

A study has been carried out in Argentina on samples of corn genotypes from a breeding station as well as in commercially available corn meal. All samples were analyzed for fungal infection and aflatoxin B₁.Mycological analysis of corn genotypes showed the presence of three principal genera of filamentous fungi Fusarium (100%), Penicillium (67%) and Aspergillus (60%). In the genus Fusarium three species were identified, F. moniliforme (42%), F. nygamai (56%) andF. proliferatum (1.8%). Eight species ofPenicillium were identified, the predominant species isolated were P. minioluteum, P. funiculosum and P. variabile. In the genus ranked third in isolation frequency, two species were identified, A. flavus and A. parasiticus, the percentage of infection was 78% and 21%, respectively. Only one corn genotype was contaminated with aflatoxin B₁ at a level of 5 ppb. The cornmeal samples showed great differences in fungal contamination, the values ranging from 1 × 10¹ to 7 × 10⁵ cfu g^–1. Fusarium (68%), Aspergillus (35%) and Penicillium (21%) were the most frequent genera isolated. Among the genus, Aspergillus, A. parasiticus (38%) was the most frequent species isolated. All the samples of corn meal were negative to aflatoxin B₁. These results indicate a low degree of human exposure to aflatoxins in Argentina through the ingestion of maize or corn meal.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mating Type Sequences in Asexually Reproducing Fusarium Species

To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative α and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The α box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.     

Prevalence and distribution of airborne and waterborne fungi and actinomycetes in the Nile river

The objective of this research was to investigate the prevalence and distribution of airborne and waterborne fungi and actinomycetes along the main stream of the Nile river during April to July, 2005. Air and water samples were collected at eight sites within a ~50 km stretch of the river. The distribution and prevalence of air and water microorganisms varied with location. The highest counts of airborne fungi (516 CFU/p/h) and actinomycetes (222 CFU/p/h) were detected at suburban sites near cultivated areas. However, the highest counts of waterborne fungi (56.4 CFU/ml) and actinomycetes (15.4 CFU/ml) were detected at Al-Galaa (city centre) and Kafr-El-elwe (south Cairo), respectively. A total of 1,816 fungal colonies (943 isolates from air and 873 from water samples) belonging to 27 genera were identified. Aspergillus, Alternaria, Cladosporium, and yeasts were the predominant fungal types in both air and water environments. Dreschlera, Emericella, Nigrospora, Spicaria, Stachybotrys, and Verticillium were only detected in the air, and Epicoccum, Philaphora, Phoma and Ulocladium were only detected in the water. Mycotoxin-producing fungi represented by Aspergillus flavus, Aspergillus parasiticus, Penicillium, Fusarium, and Trichoderma were found in the air and water environments. Significant differences (P ≤ 0.05) were found between fungal populations in air and water at different sampling sites. No significant differences (P ≥ 0.05) were found between waterborne actinomycetes. Sampling location, human activity, and pollution load are the main factors affecting the variability and biodiversity of microorganisms in different microenvironments.