The challenge for identifying the fungi living inside mushrooms: the case of truffle inhabiting mycelia

The Tuber ascomata (truffles) are a microhabitat for bacteria, viruses, and fungi (yeasts and filamentous fungi). In this survey, we tried to develop a method that would make it possible to define the mycobiome of the truffle-inhabiting filamentous fungi using culture independent molecular methods. The nested quantitative Real-Time PCR allowed us to demonstrate that each truffle is home to multiple species of filamentous fungi and that their DNA is present within the healthy ascoma at the ratio of 10^?6 compared to that of the truffle. Probably due to their insignificant presence, Denaturing Gradient Gel Electrophoresis of the amplification of ITS amplicons showed only those of the host. Based on the results, the possibilities of being able to detect the fungicolous fungi present in very small amounts within a fungal host are discussed.     

Cyanobacteria inhabiting biological soil crusts of a polar desert: Sør Rondane Mountains,Antarctica

Molecular and morphological methods were applied to study cyanobacterial community composition in biological soil crusts (BSCs) from four areas (two nunataks and two ridges) in the Sør Rondane Mountains, Antarctica. The sampling sites serve as control areas for open top chambers (OTCs) that were put in place in 2010 at the time of sample collection and will be compared with BSC samples taken from the OTCs in the future. Cyanobacterial cell biovolume was estimated using epifluorescence microscopy, which revealed the dominance of filamentous cyanobacteria in all studied sites except the Utsteinen ridge, where unicellular cyanobacteria were the most abundant. Cyanobacterial diversity was studied by a combination of molecular fingerprinting methods based on the 16S rRNA gene (denaturing gradient gel electrophoresis (DGGE) and 454 pyrosequencing) using cyanobacteria-specific primers. The number of DGGE sequences obtained per site was variable and, therefore, a high-throughput method was subsequently employed to improve the diversity coverage. Consistent with previous surveys in Antarctica, both methods showed that filamentous cyanobacteria, such as Leptolyngbya sp., Phormidium sp. and Microcoleus sp., were dominant in the studied sites.In addition, the studied localities differed in substrate type, climatic conditions and soil parameters, which probably resulted in differences in cyanobacterial community composition. Furthermore, the BSC growing on gneiss pebbles had lower cyanobacterial abundances than BSCs associated with granitic substrates.     

用于分析土壤微生物结构变化规律的变性梯度凝胶电泳条件研究

研究确定土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为进一步研究分析土壤中微生物结构变化规律提供理论依据。土壤微生物基因组DNA提取采用直接法和间接法进行比较; PCR扩增条件调整扩增体系、DGGE电泳条件调整变性剂范围,并对其结果进行比较分析。通过对DGGE电泳相关条件的研究,结果显示,土壤中粗基因组DNA采用直接法提取,然后进行纯化; PCR扩增体系中加入BSA,DGGE电泳系统组成中变性剂浓度范围为35%～55%。确定了土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为后续的相关研究提供理论依据。     

Bacterial communities associated with honeybee food stores are correlated with land use

Microbial communities, associated with almost all metazoans, can be inherited from the environment. Although the honeybee (Apis mellifera L.) gut microbiome is well documented, studies of the gut focus on just a small component of the bee microbiome. Other key areas such as the comb, propolis, honey, and stored pollen (bee bread) are poorly understood. Furthermore, little is known about the relationship between the pollinator microbiome and its environment. Here we present a study of the bee bread microbiome and its relationship with land use. We estimated bacterial community composition using both Illumina MiSeq DNA sequencing and denaturing gradient gel electrophoresis (DGGE). Illumina was used to gain a deeper understanding of precise species diversity across samples. DGGE was used on a larger number of samples where the costs of MiSeq had become prohibitive and therefore allowed us to study a greater number of bee breads across broader geographical axes. The former demonstrates bee bread comprises, on average, 13 distinct bacterial phyla; Bacteroidetes, Firmicutes, Alpha‐proteobacteria, Beta‐proteobacteria, and Gamma‐proteobacteria were the five most abundant. The most common genera were Pseudomonas, Arsenophonus, Lactobacillus, Erwinia, and Acinetobacter. DGGE data show bacterial community composition and diversity varied spatially and temporally both within and between hives. Land use data were obtained from the 2007 Countryside Survey. Certain habitats, such as improved grasslands, are associated with low diversity bee breads, meaning that these environments may be poor sources of bee‐associated bacteria. Decreased bee bread bacterial diversity may result in reduced function within hives. Although the dispersal of microbes is ubiquitous, this study has demonstrated landscape‐level effects on microbial community composition.     

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis

Aim: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). Methods: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT‐PCR‐DGGE and qPCR analysis of the bacterial and fungal communities was carried out. Results: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. Conclusions: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4°C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.     

PCR–DGGE‐based methodologies to assess diversity and dynamics of Aeromonas communities

Aims: Aeromonas is ubiquitous in aquatic environments and may cause infectious diseases in fish and humans. However, reliable and specific methods to evaluate the diversity and dynamics of Aeromonas populations are currently unavailable. This study aimed to develop PCR–DGGE methodologies for culture‐independent analysis of Aeromonas populations in water systems. Methods and Results: Three primer sets were designed to amplify selected sections of genes gyrB, rpoD and sodB from Aeromonas. Their specificity was confirmed by in silico analysis and by PCR on DNA from pure cultures. Estuarine water samples were analyzed by PCR–DGGE using those primers. DGGE patterns clearly clustered according to seasonal factors, and Aeromonas communities were surprisingly stable along a salinity gradient. Sequences of cloned amplicons affiliated to sequences belonging to seven Aeromonas species previously isolated from the same environment. Conclusions: The three systems used showed to be useful to describe the diversity of Aeromonas communities. However, the combined use of more than one primer set is advisable. Significance and Impact of the Study: The methods presented here can be applied to understand the natural pool of Aeromonas and also to monitor and control these bacteria in aquatic reservoirs.     

Microbial community interactions and population dynamics of an algicidal bacterium active against Karenia brevis (Dinophyceae)

The population dynamics of Cytophaga strain 41-DBG2, a bacterium algicidal to the harmful algal bloom (HAB) dinoflagellate Karenia brevis, were investigated in laboratory experiments using fluorescent in-situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Following its introduction into non-axenic K. brevis cultures at concentrations of 10³ or 10⁵ bacterial cells per milliliter, 41-DBG2 increased to 10⁶ cells per milliliter before initiation of its algicidal activity. Such threshold concentrations were not achieved when starting algal cell numbers were relatively low (10³ cells per milliliter), suggesting that the growth of this bacterium may require high levels of dissolved organic matter (DOM) excreted by the algae. It remains to be determined whether this threshold concentration is required to trigger an algicidal response by 41-DBG2 or, alternatively, is the point at which the bacterium accumulates to an effective killing concentration. The ambient microbial community associated with these algal cultures, as determined by DGGE profiles, did not change until after K. brevis cells were in the process of lysing, indicating a response to the rapid input of algal-derived organic matter. Resistance to algicidal attack exhibited by several K. brevis clones was found to result from the inhibition of 41-DBG2 growth in the presence of currently unculturable bacteria associated with those clones. These bacteria apparently prevented 41-DBG2 from reaching the threshold concentration required for initiation of algicidal activity. Remarkably, resistance and susceptibility to the algicidal activity of 41-DBG2 could be transferred between K. brevis clones with the exchange of their respective unattached bacterial communities, which included several dominant phylotypes belonging to the α-proteobacteria, γ-proteobacteria, and Cytophaga–Flavobacterium–Bacteroides (CFB) groups. We hypothesize that CFB bacteria may be successfully competing with 41-DBG2 (also a member of the CFB) for nutrients, thereby inhibiting growth of the latter and indirectly providing resistance against algicidal attack. We conclude that if algicidal bacteria play a significant role in regulating HAB dynamics, as some authors have inferred, bacterial community interactions are crucial factors that must be taken into consideration in future studies.     

Natural populations of the entomopathogenic fungus Beauveria bassiana in Chinese forest ecosystems are diverse and reveal equal frequencies of mating types within phylogenetic species

Beauveria bassiana is an important entomopathogenic fungus with widespread application in the biological control of harmful insect pests. This species is widely distributed as an anamorph while only two teleomorph specimens have been found in eastern China. However, little is known about the ecological conditions for sexual reproduction in natural populations of B. bassiana. Here, we collected 488 isolates of Chinese B. bassiana sensu stricto from five sites, in which teleomorph or anamorph occurred, and used molecular phylogenetic and haplotype information to determine phylogenetic diversity, mating types, and sexual reproductive potential in these populations. Molecular identification based on denaturing gradient gel electrophoresis (DGGE) and combined data of the nuclear intergenic region Bloc and translation elongation factor-1a (TEF) assemblage resolved five B. bassiana s.s phylogenetic species labeled according to their geographic origin: Europe/N. Africa 1, Asia 3, Asia 4, AFNEO_1, and N. America 2. In Guniujiang and Manshuihe collection sites, teleomorph isolates RCEF 0771 and RCEF 0382 were both identified as Europe/N. Africa 1 phylogenetic species. In addition, more than half of the isolates in five representative sites belonged to Europe/N. Africa 1. However, the teleomorph of B. bassiana s.s. was not detected in Kuankuoshui while isolates within Europe/N. Africa 1 were present at this site, and isolates belonging to Europe/N. Africa 1 were not found in either Jingyuetan or Dinghushan collection sites. Distribution of MAT1 and MAT2 mating type idiomorphs in Europe/N. Africa 1 were 51:69, 37:24, and 15:15 in Guniujiang, Manshuihe, and Kuankuoshui, respectively. The presence of teleomorph and roughly equal frequencies of opposite mating types indicate regular sexual reproduction in B. bassiana natural populations. The data offer a better understanding of the ecological conditions of sexual reproduction in natural populations of B. bassiana. These results also yield insights into the potential for sexual reproduction in other supposedly ‘asexual’ fungi.     

A bacterial population study of commercialized wastewater inoculants

AIMS: To assess the bacterial diversity and safety of wastewater inoculants, which are commercially available products used to improve the aerobic digestion processes of the domestic waste compost in the septic tank. METHODS AND RESULTS: Eighteen wastewater inoculants were analysed on nonselective and selective media and the cultivable bacteria were identified. In all wastewater inoculants, the number of CFUs were between 10(4) and 10(7) g(-1) powder on nonselective media and Bacillus was the predominant cultivable genus. Culture-independent molecular methods such as sequencing of 16S rRNA clone libraries and denaturating gradient gel electrophoresis demonstrated the high prevalence of interfering chloroplast 16S rRNA from plant material and the presence of Bacillus spp. Only after selective enrichments and cultivation, the presence of one pathogenic strain (Klebsiella pneumoniae subsp. pneumoniae) and one opportunistic strain of (Enterobacter cloacae) bacteria were detected in six different products. CONCLUSION: The predominant cultivable species of the wastewater inoculants were Bacillus spp. and after enrichment six products were found to contain opportunistic or pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of opportunistic pathogenic strains in the inoculants might represent a risk for immunocompromised, the elderly or children. A clear labelling should therefore be displayed on the product.     

Changes in arbuscular mycorrhizal fungal communities during invasion by an exotic invasive plant

Exotic invasive plants can show strong plant–soil feedback responses, but little is known about time scales for significant changes in soil microbial communities to occur after invasion. Previous work has suggested that plant invasions can modify arbuscular mycorrhizal (AM) fungal community structure. However, there is a lack of understanding about how long it takes for these changes to develop. To test this we investigated temporal changes in AM fungal communities colonising the invasive plant Vincetoxicum rossicum (Apocynaceae). We hypothesised that AM fungal community structure would change in a particular direction during the invasion process. We collected soil from two sites with a long history of invasion by this plant, with each site having paired invaded and uninvaded plots. Soil from these plots was used in a glasshouse experiment to characterise AM fungal community structure in the roots of V. rossicum at different times throughout a simulated growing season. AM fungal community structure differed between invaded and uninvaded plots. However, contrasting with our hypothesis, AM fungal communities colonising V. rossicum growing in soil from uninvaded plots did not change towards those in plants growing in previously invaded soil. Our data suggest that changes to AM fungal communities in the presence of V. rossicum require longer than the first growing season after establishment to develop.