Detection and identification of fungal species by electronic nose technology: A systematic review

A rapid and effective identification of fungal species is essential for numerous applications, and electronic nose systems are being proposed as suitable alternatives to currently available fungi identification techniques. Hence, the present review aims to unveil all published information concerning fungi identification by electronic nose systems.A systematic review of the literature was conducted according to the PRISMA guidelines. A total of 16 articles met the inclusion criteria and were included in the analysis. The results of the reviewed studies demonstrated that effective detection of fungi was possible through sensor-based electronic nose systems, which may actually function as a mycotoxin screening tool for several applications.The obtained results suggest that the sensor-based electronic nose systems may not only screen different fungi genera, but also identify the associated species. This technology has already been experimented in several fields, from food industry to clinical practice.By summarizing these results, the present review may accelerate the standardization of electronic noses in fungi detection and discrimination, allowing a faster and more efficient screening of samples.     

Biodeterioration of Mayan archaeological sites in the Yucatan Peninsula, Mexico

Many of the monuments of the Mayan civilization are suffering deterioration caused by environmental factors (high temperatures and relative humidities), increasing contamination from natural and anthropogenic sources, and by the action of micro- and macro-biological communities. Archaeological sites and historical monuments in the Mayan area were constructed with different limestones which offer different resistances to degradation by the various types of contamination. Two different sampling sites were chosen at the archaeological site of Uxmal in the Yucatan Peninsula, Mexico. Heterotrophic bacteria, cyanobacteria and different fungi were isolated and classified taxonomically. The other archaeological site chosen for this study was the fortress of Tulum, located at the side of the Caribbean Sea and exposed to chloride of marine spray and sand erosion. In this case, heterotrophic aerobic and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling areas selected. In both archaeological sites crust deposits were observed by using light microscopy, SEM and ESEM. Surface analyses were made by means of EDAX and electron microprobe. Possible mechanisms of stone decay, based on the type of microorganisms isolated, the physico-chemical characteristics of the constructional materials and environmental factors are discussed.     

Diagnosis of acute puerperal metritis by electronic nose device analysis of vaginal discharge in dairy cows

The objective of this study was to estimate the diagnostic accuracy of an electronic nose device using vaginal discharge samples to diagnose acute puerperal metritis (APM) in dairy cows. Uterine fluid was sampled manually with a gloved hand and under sterile conditions for electronic nose device analysis (day in milk (DIM) 2, 5, and 10) and bacteriologic examination (DIM 5), respectively, and on additional days, if APM was diagnosed during the daily clinical examinations. A dataset containing samples from 70 cows was used to create a model and to validate the APM status predicted by this model, respectively. Half of the dataset (n = 35; 14 healthy and 21 metritic cows) was provided with information regarding the APM diagnosis and contained all three measurements (DIM 2, 5, and 10) for each cow and was used as a training set whereas the second half was blinded (n = 35; 14 healthy and 21 metritic cows) and contained only the samples collected on DIM 5 of each cow and was used to validate the created prediction model. A receiver operating characteristic curve was calculated using the prediction results of the validation test. The best observed sensitivity was 100% with specificity of 91.6% when using a threshold value of 0.3. The calculated P-value for the receiver operating characteristic curve was less than 0.01. Overall, Escherichia coli was isolated in eight of 28 (28.6%) and 22 of 42 (52.4%) samples collected from healthy and metritic cows, respectively. Trueperella pyogenes and Fusobacterium necrophorum were isolated in 14 and six of 28 (50.0% and 21.4%) and 17 and 16 of 42 (40.5% and 38.1%) samples collected from healthy and metritic cows, respectively. The prevalence of Escherichia coli and Trueperella pyogenes was similar in the samples obtained from metritic cows used for the training set and the validation test. The results are promising especially because of the objective nature of the measurements obtained by the electronic nose device.     

Evolving challenges and strategies for fungal control in the food supply chain

Fungi that spoil foods or infect crops can have major socioeconomic impacts, posing threats to food security. The strategies needed to manage these fungi are evolving, given the growing incidence of fungicide resistance, tightening regulations of chemicals use and market trends imposing new food-preservation challenges. For example, alternative methods for crop protection such as RNA-based fungicides, biocontrol, or stimulation of natural plant defences may lessen concerns like environmental toxicity of chemical fungicides. There is renewed focus on natural product preservatives and fungicides, which can bypass regulations for ‘clean label’ food products. These require investment to find effective, safe activities within complex mixtures such as plant extracts. Alternatively, physical measures may be one key for fungal control, such as polymer materials which passively resist attachment and colonization by fungi. Reducing or replacing traditional chlorine treatments (e.g. of post-harvest produce) is desirable to limit formation of disinfection by-products. In addition, the current growth in lower sugar food products can alter metabolic routing of carbon utilization in spoilage yeasts, with implications for efficacy of food preservatives acting via metabolism. The use of preservative or fungicide combinations, while involving more than one chemical, can reduce total chemicals usage where these act synergistically. Such approaches might also help target different subpopulations within heteroresistant fungal populations. These approaches are discussed in the context of current challenges for food preservation, focussing on pre-harvest fungal control, fresh produce and stored food preservation. Several strategies show growing potential for mitigating or reversing the risks posed by fungi in the food supply chain.     

电子圆二色谱技术在微生物次级代谢产物结构研究中的应用研究进展

微生物次级代谢产物的化学结构十分复杂,对其绝对构型的确定十分困难。近年来,电子圆二色谱(electronic circular dichroism,ECD)由于其用量少、精度高等优点,在测定绝对构型方面的应用越来越多,已经成为研究微生物次级代谢产物结构的重要方法。本文就电子圆二色谱在微生物次级代谢产物结构研究中的应用进行综述,以期为今后的研究奠定基础。     

Sensor systems,electronic tongues and electronic noses,for the monitoring of biotechnological processes

Production of biofuel is based on the conversion by microorganisms of complex organic substrates into the methane or ethanol, which are consequently used as energy sources. Real time monitoring of the fermented media composition is of paramount for the effectiveness of the whole process. However, despite the fact that products worth billions of dollars are produced through fermentation processes annually, analytical instruments used for these processes’ monitoring remain relatively primitive. Established laboratory techniques produce exhaustive information about media composition but analysis is often quite time-consuming, expensive, requires skilled personnel and hardly can be automated. Lack of on-line sensors for the fermentation monitoring is commonly stressed in the literature. One of the techniques particularly suitable for this purpose is chemical sensors. Such features as low prices, relatively simple instrumentation, minimal sample preparation and easy automation of measurements make chemical sensors an attractive tool for industrial process control. However, practical use of chemical sensors in complex media is often hindered by their insufficient selectivity. For example, only pH and oxygen probes are routinely used in bio-reactors. One of the emerging approaches permitting to overcome the selectivity problems is the use of systems instead of discrete sensors. Such systems for liquid and gas analysis were named electronic tongues and electronic noses correspondingly. They are capable to perform both quantitative analysis (components’ concentrations) and classification or recognition of multicomponent media. This review presents recent achievements in the R&D and applications of electronic tongues and noses to the monitoring of biotechnological processes. JIMB-2008: BioEnergy—Special issue.     

Potential for detection of microorganisms and heavy metals in potable water using electronic nose technology

Studies have been carried out to determine the potential for the detection of different microbial species (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa), alone and in the presence of low concentrations of different heavy metals (As, Cd, Pb and Zn) in bottled, reverse osmosis (RO) and tap water, using an electronic nose. Studies show that it is possible to discriminate control water samples from water contaminated with 0.5 ppm of a mixture of metals. The presence of heavy metals may modify the activity of microorganisms and thus the volatile production patterns. Bacterial species at 10(2)-10(4) colony forming units (CFUs) ml(-1) could be detected after 24 h of incubation. Work is in progress to identify the limits of detection for a range of other microorganisms, including, fungi and cyanobacteria, and chlorinated phenols using electronic nose technology.     

Microbial and enzymatic control of pitch in the pulp and paper industry

Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching.     

Early detection of bacterial diseases in apple plants by analysis of volatile organic compounds profiles and use of electronic nose

DNA‐based protocols are the standard methods for the diagnosis of infected plant material. Nevertheless, these methods are time‐consuming and require trained personnel, with an efficacy depending on the sampling procedure. In comparison, recognition methods based on volatile compounds emissions are less precise, but allow a non‐destructive mass screening of bulk samples, and may be implemented to steer molecular diagnosis. In this study, the analysis of volatile compounds is used for the discrimination of fire blight (Erwinia amylovora) and blossom blight (Pseudomonas syringae pv. syringae) on apple propagation material. Possible marker compounds were identified by gas chromatography–mass spectroscopy (GC‐MS) and proton transfer reaction‐time of flight‐mass spectroscopy (PTR‐ToF‐MS). In addition, two commercial electronic noses were used for diagnosis. After a preliminary validation in vitro, a diagnostic protocol was successfully developed to scale up to real nursery conditions on cold stored, asymptomatic dormant plants.     

Advances in fundamental and applied studies in China of fungal biocontrol agents for use against arthropod pests

Entomopathogenic fungi, such as Beauveria bassiana and Metarhizium anisopliae, are environmentally friendly biocontrol agents (BCAs) against various arthropod pests. We provide an overview to the past-decade advances in fungal BCA research and application in China. Since 1960s, fungal BCAs have been mass-produced for application and at present, thousands of tons of their formulations are annually applied to control forest, agricultural, greenhouse and grassland insect pests throughout the country. Apart from technical advances in mass production, formulation and application of fungal BCAs, basic studies on the genomics, molecular biology, genetic engineering and population genetics of fungal entomopathogens have rapidly progressed in the past few years in China. The completed genomic studies of M. anisopliae, Metarhizium acridum, B. bassiana and Cordyceps militaris provide profound insights into crucial gene functions, fungal pathogenesis, host–pathogen interactions and mechanisms involved in fungal sexuality. New knowledge gained from the basic studies has been applied to improve fungal virulence and stress tolerance for developing more efficacious and field-persistent mycoinsecticides by means of microbial biotechnology, such as genetic engineering. To alleviate environmental safety concerns, more efforts are needed to generate new data not only on the effects of engineered BCAs on target and non-target arthropods but also on their potential effects on gene flow and genetic recombination before field release.     

Delving into the dark ecology: A continent-wide assessment of patterns of composition in soil fungal communities from Australian tussock grasslands

Soil fungi play essential roles in many terrestrial processes, but our knowledge of the forces governing fungal distribution and community composition along broad-scale environmental gradients is still limited. In this study, we explored biogeographic distribution and composition of soil fungal communities associated with 62 tussock grasslands across different regions of Australia. Climatic parameters had only a limited correlation with fungal community structure, while edaphic variables and spatial distance were significantly associated with changes in fungal community composition. We also observed high variations in composition among fungal assemblages from different ecological regions, suggesting some regional endemism in these communities. The discrete distribution of fungi in soil was further confirmed by indicator analysis, which identified distinct indicator operational taxonomic units associated with grasslands from different climatic regions. Finally, fungi with flexible trophic interactions had a central role in the network architecture of both arid and temperate communities. Taken together, the results from our study confirm the prominent role of soil physico-chemical status and geographic location in determining fungal biogeographic patterns over large scales in Australia.     

Application of image analysis in the fungal fermentation of Trichoderma reesei RUT-C30

Trichoderma reesei was grown in a stirred-tank bioreactor (STB) and a reciprocating plate bioreactor (RPB) at four different agitation speeds. A semiautomatic image analysis protocol that was developed to characterize the mycelium morphology during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms) was applied to study the effect of agitation on the morphology of T. reesei. It was shown via statistical validation that broth samples used for image analysis represented the whole population of the fungi in the bioreactor. High shear was found to be damaging to T. reesei grown in the STB. The gentler shear produced in the RPB was not detrimental to the microorganism even at higher agitation speed. Better productivity was obtained for T. reesei grown in the STB and the highest productivity, 0.121 IU/mL h, was obtained at 400 rpm. The morphological parameter, the hyphal growth unit, was found to be correlated to the productivity. Understanding the effect of agitation on the morphology and productivity of T. reesei could lead to the design of better bioreactors and the selection of operating conditions of bioreactors to optimize the production of cellulase.     

Application of species richness estimators for the assessment of fungal diversity

Species richness and distribution patterns of wood-inhabiting fungi and mycetozoans (slime moulds) were investigated in the canopy of a Central European temperate mixed deciduous forest. Species richness was described with diversity indices and species-accumulation curves. Nonmetrical multidimensional scaling was used to assess fungal species composition on different tree species. Different species richness estimators were used to extrapolate species richness beyond our own data. The reliability of the abundance-based coverage estimator, Chao, Jackknife and other estimators of species richness was evaluated for mycological surveys. While the species-accumulation curve of mycetozoans came close to saturation, that of wood-inhabiting fungi was continuously rising. The Chao 2 richness estimator was considered most appropriate to predict the number of species at the investigation site if sampling were continued. Gray's predictor of species richness should be used if statements of the number of species in larger areas are required. Multivariate analysis revealed the importance of different tree species for the conservation and maintenance of fungal diversity within forests, because each tree species possessed a characteristic fungal community. The described mathematical approaches of estimating species richness possess great potential to address fungal diversity on a regional, national, and global scale.     

Application of beneficial rhizospheric microbes for the mitigation of seed-borne mycotoxigenic fungal infection and mycotoxins in maize

In the present investigation, seven rhizobacteria and nine Trichoderma spp. were evaluated to suppress seed-borne mycotoxigenic fungi (Aspergillus flavus and Fusarium verticillioides) and mycotoxin (aflatoxin and fumonisin) and to improve planting value of maize. Under in vitro conditions, these beneficial microorganisms suppressed the growth of A. flavus and F. verticillioides to various extents. Bacillus sp. (Bsp 3/aM), Pseudomonas putida (Has 1/c), Trichoderma asperellum (M5) and T. asperellum (T2) exhibited the greatest antagonistic effect on seed-borne mycotoxigenic fungi, and subsequently reduced mycotoxin concentrations in seeds. Under greenhouse conditions, these four biocontrol strains were also found to increase root length, shoot length, % germination, vigour index, fresh weight and dry weight of seedlings. Considering their overall performances, strains Bsp 3/aM, Has 1/c, M5 and T2 were selected for field studies as microbial talcum formulations. Among the tested microbial formulations, strain Bsp 3/aM significantly increased yield by 9.4% and 6.2% over the control in two maize cultivars Hema and Pearl, respectively. Increased plant growth and yield was also correlated with nutrient uptake in both the tested cultivars. All microbial formulation recorded significantly (p ≤ 0.05) reduced A. flavus infection and aflatoxin contamination in harvested seeds. But, none of the microbial formulations were found significant (p ≤ 0.05) in reducing F. verticilliodes incidence and fumonisin contamination. Our findings indicate that these microbial antagonists indirectly improve host health by suppressing seed-borne incidence of mycotoxigenic fungi and directly by facilitating nutrient uptake, thereby revealing their potential as both biofertilisers and biopesticides for maize production.     

Application of PCR for detection and identification of mycoplasma contamination in virus stocks

Summary A nested polymerase chain reaction (PCR) was used to detect and identify mycoplasma contaminants in viral stocks. The results of the PCR assay proved to be a sensitive and accurate indicator of the true status of the stock tested. Those samples positive by agar culture or Hoechst stain were also positive by PCR. Those samples that were inconclusive by Hoechst stain (10.05%) could be clearly determined to be mycoplasma positive or negative by PCR. The PCR assay also detected those fastidious species of mycoplasma that gave false negative results by the direct culture method. In many respects the PCR-based mycoplasma detection method described is superior to the agar culture and Hoechst staining detection methods. In this study, the PCR assay detected substantially more mycoplasma-positive viral stocks than did the agar culture assay. Due to its speed, sensitivity, and reliability, the PCR assay is of particular value in monitoring the process of removing mycoplasma from contaminated stocks. Furthermore, the PCR amplification products can be analyzed by restriction analysis to rapidly identify the species of the mycoplasma contaminating the stock tested.     

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.