Axenic Medium Research Articles

Morphological, molecular and pathogenic diagnostic of fungi in lawn’s root and neck in Mexico

The illnesses that cause root and neck rot in golf courses constitute a limiting factor for grass development worldwide. This is mainly true when management guidelines are not conducted. Our research was conducted in the Golf Club “Mexico”, located in Mexico, D.F. Isolates were taken from plants that presented yellowing symptoms and root and neck rot. The identified pathogenic fungi, isolated from axenic media, were Giberella avenacea, Fusarium oxysporum, F. oxysporum f. sp. cubense and Pythium ultimum. The molecular characterization showed a comparison of the DNA-obtained sequences which was morphologically determined as F. avenaceum and was molecularly corroborated as G. avenacea with a similarity of 96%. Comparison between the obtained DNA sequences which were morphologically determined as F. oxysporum, were molecularly corroborated as F. oxysporum with a similarity of 97%. Finally, the DNA fungi sequence comparisons that were morphologically determined as F. oxysporum, were molecularly corroborated as F. oxysporum f. sp. cubense with a similarity of 97%. It was proved that the study fungi were the causal agents of the symptoms at the field because their inoculation in grass growing in plots (at the greenhouse) determined the same symptoms. The re-isolates were identified as the inoculated species, and in this way the Koch’s postulates were fulfilled.

Beginning to Understand the Role of the Type IV Secretion System Effector Proteins in Coxiella burnetii Pathogenesis.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which manifests in severe outbreaks and is associated with important health and economic burden. Moreover, C.burnetii belongs to the list of class B bioterrorism organisms, as it is an airborne and highly infective pathogen with remarkable resistance to environmental stresses. Detailed study of the host-pathogen interaction during C. burnetii infection has been hampered due to the obligate intracellular nature of this pathogen. However, the development of an axenic culture medium, together with the implementation of bioinformatics tools and high-content screening approaches, have significantly progressed C. burnetii research in the last decade. This has facilitated identification of the Dot/Icm type IV secretion system (T4SS) as an essential virulence factor. T4SS is used to deliver an arsenal of effector proteins into the cytoplasm of the host cell. These effectors mediate the survival of the host cell and the development of very large replicative compartments called Coxiella-containing vacuoles (CCVs). Biogenesis of the CCV relies on T4SS-dependent re-routing of numerous intracellular trafficking pathways to deliver membranes and nutrients that are essential for bacterial replication. This review aims to illustrate the key milestones that have contributed to ascribe C. burnetii as a model organism for the study of host/pathogen interactions as well as presenting an up-to-date description of our knowledge of the cell biology of C. burnetii infections.

Oligosaccharides containing an α-(1 → 2) (glucosyl/xylosyl)-fructosyl linkage as inducer molecules of trichothecene biosynthesis for Fusarium graminearum

Fructo-oligosaccharides containing a sucrose unit are reported as carbon sources necessary for trichothecene production by Fusarium graminearum. Here we demonstrate that trichothecene production is induced when at least 100μM sucrose is added to a culture medium containing 333mM glucose in a 24-well plate. When glucose, the main carbon source of the medium, was replaced with galactose, maltose, or sorbitol, the addition of 100μM sucrose could no longer induce trichothecene production. However, replacing half the amount of each carbon source with glucose restored the trichothecene production-inducing activity of sucrose. Detailed investigations with media containing various concentrations of galactose and glucose as carbon sources suggested that operation of the galactose catabolic pathway for energy conservation affected trichothecene biosynthesis induction by sucrose. Trichothecene production was also induced by 100μM of either raffinose or xylosucrose in axenic liquid culture medium containing glucose as the major carbon source. These results demonstrate that sucrose derivatives are not necessary as a carbon source for inducing trichothecene biosynthesis, and that the minimum structural requirement for sugars to function as trichothecene production-inducer molecules is to contain an α-(1→2) (glucosyl/xylosyl)-fructosyl linkage.

Investigation of potentially pathogenic free-living amoebae and their in vivo pathogenicity in water supplies of Turkey

Free-living amoebae (FLA) are found widely in soil and water in the nature. Among them in which potentially pathogenic for humans and animals are known as "potential pathogenic free-living amoebae (PPFLA)". PPFLA are characterized as the causes of clinical manifestations leading to death especially in immunosuppressed people. Four genus of PPFLA (Acanthamoeba, Naegleria, Balamuthia and Sappinia) are known to be pathogenic to humans. The aims of this study were to investigate the presence of PPFLA in the water supplies in Turkey and to determine their in vivo pathogenicity. A total of 664 water samples were collected from the ponds, rivers, streams and wells found in provinces located at different regions (central, western, eastern and southeastern regions) of Turkey. These samples were initially inoculated in the monoxenic culture media and evaluated by both microscopy and polymerase chain reaction (PCR) in terms of the presence of FLA. The samples identified as positive were then cultured in axenic media, the growth of amoebae that were confirmed microscopically, were than studied with PCR for molecular characterization. The isolates that were found positive by PCR from axenic cultures were inoculated intranasally to immunocompetent and immunodeficient (athymic) [BALB/c Rag2(-/-) gamma(c)(-/-)] BALB/c mice followed by the evaluation on the 21st day by histopathological and molecular methods to investigate their in vivo pathogenicity. In our study, 143 water samples were detected as positive in monoxenic cultures and 41 of them were detected as positive in axenic cultures. Twenty of 41 samples detected as positive in axenic culture could be continued in culture for three months. As a result of PCR using primers common to SYA, only nine have been identified from 20 samples as positive. According to the result of the PCR with specific primers, all (n= 9) were positive for Acanthamoeba sp., eight for Sappini sp. and five for Balamuthia mandrillaris, while none was observed Naegleria fowleri. Histopathologic examination revealed that both groups of mice that were infected with the nine isolates had normal brain tissue sections; but haemorrhages and mononuclear cell proliferation were determined in four immunocompetent and seven athymic animal lung sections. When the presence of parasites in tissue samples were evaluated by real-time PCR, Balamuthia was detected in at least one blood, lung, brain or nasal mucosa sample of the four immunocompetent mice, Sappinia sp. in four and Acanthamoeba sp. in seven immunocompetent mice infected with nine isolates. Additionally, seven Balamuthia sp., seven Sappinia sp. and eight Acanthamoeba sp. were detected in immunodeficient mice. In this study, B. mandrillaris and Sappinia sp. were the first isolated potentially pathogenic amoebae from water supplies located at different parts of Turkey. As a result awareness and precautions against suspicious water supplies used for drinking, daily use and swimming purposes should be treated more carefully.

Genotyping and Axenic Growth of Coxiella burnetii Isolates Found in the United States Environment.

Coxiella burnetii is a gram-negative bacterium that is the etiologic agent of the zoonotic disease Q fever. Common reservoirs of C. burnetii include sheep, goats, and cattle. These animals shed C. burnetii into the environment, and humans are infected by inhalation of aerosols. A survey of 1622 environmental samples taken across the United States in 2006-2008 found that 23.8% of the samples contained C. burnetii DNA. To identify the strains circulating in the U.S. environment, DNA from these environmental samples was genotyped using an SNP-based approach to derive sequence types (ST) that are also compatible with multispacer sequence typing methods. Three different sequence types were observed in 31 samples taken from 19 locations. ST8 was associated with goats and ST20 with dairy cattle. ST16/26 was detected in locations with exposure to various animals and also in locations with no direct animal contact. Viable isolates were obtained for all three sequence types, but only the ST20 and ST16/26 isolates grew in acidified citrate cysteine medium (ACCM)-2 axenic media. Examination of a variety of isolates with different sequence types showed that ST8 and closely related isolates did not grow in ACCM-2. These results suggest that a limited number of C. burnetii sequence types are circulating in the U.S. environment and these strains have close associations with specific reservoir species. Growth in ACCM-2 may not be suitable for isolation of many C. burnetii strains.

ChIP and Chips: Introducing the WormPharm for correlative studies employing pharmacology and genome-wide analyses in C. elegans.

We present the WormPharm, an automated microfluidic platform that utilizes an axenic medium to culture C. elegans. The WormPharm is capable of sustaining C. elegans for extended periods, while recording worm development and growth with high temporal resolution ranging from seconds to minutes over several days to months. We demonstrate the utility of the device to monitor C. elegans growth in the presence of varying doses of nicotine and alcohol. Furthermore, we show that C. elegans cultured in the WormPharm are amendable for high-throughput genomic assays, i.e. chromatin-immunoprecipitation followed by next generation sequencing, and confirm that nematodes grown in monoxenic and axenic cultures exhibit genetic modifications that correlate with observed phenotypes. The WormPharm is a powerful tool for analyzing the effects of chemical, nutritional and environmental variations on organism level responses in conjunction with genome-wide changes in C. elegans.

COXIELLA BURNETII PATHOGENICITY MOLECULAR BASIS

Coxiella burnetii is an obligate intracellular gram-negative bacterial pathogen, an ethiological agent of Q-fever, a zoonotic disease, elapsing as an acute (mostly atypical pneumonia) or a chronic (mostly endocarditis) form. The host range is represented by wide range of mammal, avian and arthropod species, but the main source of human infection are farm animals. The main route of infection is aerosolic. In case of contact with organism pathogen binds with phagocytal monocytic-macrophagal cell line. C. burnetii promotes maturation of specific phagolysosome-like compartment in host cell, called coxiella-containing vacuole, within this vacuole pathogen becames metabolically activated and actively replicates. Coxiella persists as metabolically inactive spore-like form in environment. Internalisation of C. burnetii occurs using actin-mediated phagocytosis and zipper mechanism. After internalization of bacteria maturation of phagolysosome-like compartment and large coxiella-containing vacuole formation occure, and vacuole can occupy nearly the whole cytoplasm of the host cell. Survivance of infected cells is important for chronic infection with C. burnetii. C. burnetii elongate the viability of host cell by two ways: it actively inhibits apoptotic signal cascades and induce pro-survival factors. Exceptthat C. burnetii involves autophagic pathway during coxiella-containing vacuole formation, and induction of autophagy promotes pathogen replication. During infection C. burnetii translocates effector substrates from bacterial cytosole to euca ryotic host cell cytosole using type IV secretion system, where effectors modulate host cell proteins. Overall approximately 130 secreted effectors of type IV transport system, but function of most of them remains unknown to date. Specific sec reted proteins for variety of strains and isolates were identified, confirmed that certain pathotypes of C. burnetii can exist. Identification and characterization of novel virulence factors it is now possible through axenic media for C. burnetii cultivation and development of site-specific mutagenesis and other genetic technics, which is important for research of C. burnetii molecular pathogenesis.

Correction for Sandoz et al., Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium.

Volume 82, no. 10, p. 3042–3051, 2016. Page 3043, Table 1: The mM value of calcium chloride should read “0.089.” The mg/ml and mM values of sodium phosphate dibasic should read “0.10” and “0.704,” respectively. The mg/ml and mM values of sodium bicarbonate should read “0.25” and

Trypanosoma cruzi isolated from a triatomine found in one of the biggest metropolitan areas of Latin America.

To characterize Trypanosoma cruzi (TcI) isolated from a Panstrongylus megistus specimen found in one of the biggest metropolitan areas of Latin America, the relationship between the TcI group of T. cruzi and the transmission cycle in the urban environment was studied. The T. cruzi strain, Pm, was isolated in a culture medium from the evolutionary forms present in the hindgut of a live male specimen of P. megistus found in the Jabaquara subway in São Paulo City. The sample from the triatomine showed trypomastigote forms of Trypanosomatidae, which were inoculated in the peritoneum of Balb/c mice. The sample was then inoculated in Liver Infusion Tryptose medium and J774 cells for the molecular identification and characterization of the parasite. The Pm strain of T. cruzi was identified by isolation in axenic culture medium, and based on the morphology, cell infection, growth kinetics, and molecular characterization. After isolation, the protozoan was identified as T. cruzi. No parasites were detected in the peripheral blood of the animal, which can be a characteristic inherent to the strain of T. cruzi that was isolated. Cell invasion assays were performed in triplicate in the J774 cell line to confirm the invasive ability of the Pm strain and revealed amastigote forms of the parasite within macrophages. Our biological and molecular characterizations helped understand parasite-host interactions and their evolutionary history in context of the associations between vectors, ecotopes, hosts, and groups of the parasite.

The Characterization of an Adrenergic Signalling System Involved in the Encystment of the Ocular Pathogen Acanthamoeba spp.

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective β1 adrenoceptor agonist dobutamine were found to cause >90% encystment of Acanthamoeba trophozoites compared to <30% with the controls. The selective β1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by >55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α-methyl-p-tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by β1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical β adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage.

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium.

Host cell-free (axenic) culture of Coxiella burnetii in acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required for C. burnetii growth. Metabolic reconstruction of C. burnetii predicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examine C. burnetii amino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealed C. burnetii auxotrophy for 11 amino acids, including arginine. Heterologous expression of Legionella pneumophila argGH in C. burnetii permitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection of C. burnetii genetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impair C. burnetii replication. Together, these results highlight the advantages of a nutritionally defined medium in investigations of C. burnetii metabolism and the development of genetic tools. Host cell-free growth and genetic manipulation of Coxiella burnetii have revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies of C. burnetii physiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability of C. burnetii Growth studies confirmed that C. burnetii cannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in the L. pneumophila arginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies of C. burnetii metabolism and allowing non-antibiotic-based selection of C. burnetii genetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

Role of bacteria in the production and degradation of Microcystis cyanopeptides.

The freshwater cyanobacteria, Microcystis sp., commonly form large colonies with bacteria embedded in their mucilage. Positive and negative interactions between Microcystis species and their associated bacteria have been reported. However, the potential role of bacteria in the production and degradation of cyanobacterial secondary metabolites has not been investigated. In this study, a Microcystis‐associated bacterial community was isolated and added to the axenic M. aeruginosa PCC7806 liquid culture. After 3 years of cocultivation, we studied the bacterial genetic diversity adapted to the PCC7806 strain and compared the intra‐ and extracellular concentration of major cyanopeptides produced by the cyanobacterial strain under xenic and axenic conditions. Mass spectrometric analyses showed that the intracellular concentration of peptides was not affected by the presence of bacteria. Interestingly, the produced peptides were detected in the axenic media but could not be found in the xenic media. This investigation revealed that a natural bacterial community, dominated by Alpha‐proteobacteria, was able to degrade a wide panel of structurally varying cyclic cyanopeptides.

Both Free Indole-3-Acetic Acid and Photosynthetic Performance are Important Players in the Response of Medicago truncatula to Urea and Ammonium Nutrition Under Axenic Conditions.

We aimed to identify the early stress response and plant performance of Medicago truncatula growing in axenic medium with ammonium or urea as the sole source of nitrogen, with respect to nitrate-based nutrition. Biomass measurements, auxin content analyses, root system architecture (RSA) response analyses, and physiological parameters were determined. Both ammonium and ureic nutrition severely affected the RSA, resulting in changes in the main elongation rate, lateral root development, and insert position from the root base. The auxin content decreased in both urea- and ammonium-treated roots; however, only the ammonium-treated plants were affected at the shoot level. The analysis of chlorophyll a fluorescence transients showed that ammonium affected photosystem II, but urea did not impair photosynthetic activity. Superoxide dismutase isoenzymes in the plastids were moderately affected by urea and ammonium in the roots. Overall, our results showed that low N doses from different sources had no remarkable effects on M. truncatula, with the exception of the differential phenotypic root response. High doses of both ammonium and urea caused great changes in plant length, auxin contents and physiological measurements. Interesting correlations were found between the shoot auxin pool and both plant length and the “performance index” parameter, which is obtained from measurements of the kinetics of chlorophyll a fluorescence. Taken together, these data demonstrate that both the indole-3-acetic acid pool and performance index are important components of the response of M. truncatula under ammonium or urea as the sole N source.

The Contribution of Genomics to the Study of Q Fever

Coxiella burnetii is the etiological agent of Q fever, a worldwide zoonosis that can result in large outbreaks. The birth of genomics and sequencing of C. burnetii strains has revolutionized many fields of study of this infection. Accurate genotyping methods and comparative genomic analysis have enabled description of the diversity of strains around the world and their link with pathogenicity. Genomics has also permitted the development of qPCR tools and axenic culture medium, facilitating the diagnosis of Q fever. Moreover, several pathophysiological mechanisms can now be predicted and therapeutic strategies can be determined thanks to in silico genome analysis. An extensive pan-genomic analysis will allow for a comprehensive view of the clonal diversity of C. burnetii and its link with virulence.

Wormpharm: A Microfluidic Platform for Pharmacogenetic Studies on C. Elegans

Our lab has developed the WormPharm platform that utilizes microfluidics to allow automated culturing and imaging of C. elegans exposed to pharmaceuticals. Current problems with pharmaceutical experiments using C. elegans include, extensive plate maintenance and animal transfer, and additional drug metabolism by bacterial food sources. As a solution, we have modified a chemically synthetic axenic media for use with the WormPharm platform. System automation allows us to perform experiments that can last weeks to months. Alcohol, caffeine, and nicotine constitute the three most commonly consumed psychoactive drugs around the globe, making them prime compounds for pharmaceutical assays. To date, our platform has been used to study the individual and combinatorial effects of all three drugs. In our study, we found that the effects of these drugs when introduced in axenic media varied greatly both individually and in combination. We have examined locomotive behavior, pharyngeal pump rate, and morphological deformities upon drug dose. Our results show that these psychoactive drugs cause severe phenotypic effects on C. elegans when used in combination, but not individually. Additionally, the WormPharm is able to culture ample amounts of worms to conduct next generation sequencing, and we present results correlating phenotype and genotype with different combination of alcohol, caffeine and nicotine.

Anti-Giardia lamblia activity of ginger (Zingiber officinale) extract in an improved modified axenic culture

Background Giardiasis is an important common intestinal infection that occurs as a result of ingestion of cysts of the protozoan parasite Giardia lamblia. Several medications are available to treat giardiasis. Metronidazole and other chemical drugs currently used for treatment cause side effects, whereas ginger has been used for centuries as a herbal medicine, without harmful side effects. Objective With regard to the above-mentioned properties of ginger we were prompted to evaluate its anti-Giardia activity, as compared with nitazoxanide (NTZ) and phosphate buffer saline (PBS) as controls, and to establish a G. lamblia axenic culture that yields a large number of trophozoites. Materials and methods Fresh clinical isolates of G. lamblia were obtained from different patients with acute giardiasis. Trophozoites were cultured using Stone’s modification of Locke’s solution as an axenic culture medium modified by supplementing with bovine bile and heat-inactivated bovine serum. Ginger extract was prepared to give a final concentration of 20 mg/ml. In vitro assessment of effect of ginger was carried out after 24 and 48 h. For post-treatment evaluation, the viability of G. lamblia trophozoites was tested by their morphological criteria and dye staining (eosin stain 0.01%). Results The culture yielded a rich growth of G. lamblia trophozoites. Dead trophozoites stained pink with eosin and showed loss of morphological criteria. NTZ treatment significantly lowered the number of the parasites after 48 h (mean: 42.5±3.53/ml; P≤0.002), with a reduction rate of 92.93%, compared with PBS. Ginger treatment significantly lowered the number of the parasites after 48 h (mean: 55±7.07/ml, P≤0.004), with a reduction rate of 94.4%, compared with PBS. Conclusion The present study confirmed that ginger extract is equally active against G. lamblia as NTZ. More research studies are needed to highlight the physiological and molecular mechanisms of action of ginger and provide more scientific evidence of its effectiveness. Moreover, this simple G. lamblia axenic culture medium proved beneficial for evaluation of the susceptibility of isolates to antiparasitic drugs.

The cultivation of Acanthamoeba using with different axenic and monoxenic media

Objective: Acanthamoeba species are the ubiquities free-living amoebae and can infect humans, causing diseases such as keratitis and encephalitis. Acanthamoeba species are often grown on non-nutrient agar spread with Escherichia coli or peptone-yeast extract-glucose. We investigated the amount of growth of Acanthamoeba in different axenic and monoxenic media. Methods: The non-nutrient agar with Pseudomonas aeuroginosa , Enterobacter aerogenes , Staphylococcus aeurous , Escherichia coli , and Klebsiella pneumoniae were used as monoxenic media. The encystation, mycological peptone-maltose, peptone yeast extract glucose, roswell park memorial institute 1640 and trypticase beef hemoglobine media were used as axenic media. Results: We compared the growth of Acanthamoeba species in different axenic and monoxenic media in this study. In relation to the growth rate, the non-nutrient agar with Pseudomonas aeuroginosa had the highest values achieved among monoxenic media and roswell park memorial institute 1640 media was the highest value among axenic media. Conclusion: In view of the results, we can affirm that these monoxenic media are adequate to grow of Acanthamoeba species. In addition, a classic and basic medium that supports the growth of Acanthamoeba species consists of peptone yeast extract glucose. However, the roswell park memorial institute 1640 media was an excellent commercially available media for the growth of Acanthamoeba and it was able to keep Acanthamoeba by long periods of time. Key words: Acanthamoeba , Axenic, Monoxenic, Media

Rapid protocol for visualization of rust fungi structures using fluorochrome Uvitex 2B.

BackgroundHistological examination using fluorochromes is one of the standard methods for observation of microorganisms in tissues and other compartments. In the study of fungi, especially those that cannot be cultured in axenic media such as biotrophic fungi, histological examination of processes associated with the fungal growth, differentiation, infection and other cellular functions can lead to the better understanding of host-parasite interactions. Fluorescence microscopy coupled with Fluorochrome Uvitex 2B have been extensively utilized to study rust fungi structures and host-pathogen interactions. In this study, we report development of a rapid staining protocol of the rust fungus Puccinia triticina using fluorochrome Uvitex 2B. The newly developed rapid procedure was compared with a standard staining technique to observe in planta fungal infection structures development during the wheat—Puccinia triticina interaction.ResultsWhile significantly reducing the time for staining, the rapid protocol described here was equally efficient or better compared to standard procedure in detecting fungal infection structures using Uvitex 2B. In the rapid staining procedure, pre-heating of the stain increased efficiency to detect all the infection structures including haustoria with highly reduced background noise from plant tissue.ConclusionThis staining process described here is simple and quick. It can be completed in 4 h, which is of 6 times faster than the standard Uvitex 2B staining procedure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-015-0096-0) contains supplementary material, which is available to authorized users.

Acanthamoeba encystment: multifactorial effects of buffers, biocides, and demulcents present in contact lens care solutions

PurposeTo determine whether agents which are purportedly capable of inducing encystment of Acanthamoeba can recapitulate the signal when tested in differing formulations.MethodsIn accordance with the International Standard ISO 19045, Acanthamoeba castellanii ATCC 50370 trophozoites were cultured in antibiotic-free axenic medium, treated with test solutions, and encystment rates plus viability were measured via bright field and fluorescent microscopy. Test solutions included phosphate-buffered saline (PBS), borate-buffered saline, biguanide- and hydrogen peroxide (H2O2)-based biocides, propylene glycol (PG) and povidone (POV) ophthalmic demulcents, and one-step H2O2-based contact lens disinfection systems.ResultsOnly PBS solutions with 0.25 ppm polyaminopropyl biguanide (PAPB) and increasing concentrations of PG and POV stimulated A. castellanii encystment in a dose-dependent manner, whereas PBS solutions containing 3% H2O2 and increasing concentrations of PG and POV did not stimulate encystment. Borate-buffered saline and PBS/citrate solutions containing PG also did not stimulate encystment. In addition, no encystment was observed after 24 hours, 7 days, or 14 days of exposures of trophozoites to one-step H2O2 contact lens disinfection products or related solutions.ConclusionThe lack of any encystment observed when trophozoites were treated with existing or new one-step H2O2 contact lens care products, as well as when trophozoites were exposed to various related test solutions, confirms that Acanthamoeba encystment is a complex process which depends upon simultaneous contributions of multiple factors including buffers, biocides, and demulcents.

Effectiveness of a rock phosphate solubilizing fungus to increase soil solution phosphate impaired by the soil phosphate sorption capacity

Available phosphate (P) deficiency in tropical soils has been recognized as a major factor that limits soil quality and plant performance. To overcome this, it is necessary to add high amounts of soluble P-fertilizers; however, this is inefficient and costly. Alternatively, rock phosphates (RP) can be used, but their low reactivity limits their use. Phosphate solubilizing microorganisms (PSM) can enhance RP dissolution and, thus, improve the RP agronomic effectiveness as fertilizer. Nonetheless, their effectiveness may be impaired by the soil P fixation capacity. An experiment was carried out to assess the in vitro effectiveness of the fungus Mortierella sp. to dissolve RP in an axenic culture medium and, thus, enhance the solution P concentration in the presence of aliquots of soils with contrasting P fixation capacity. The results showed that the fungus was capable of lowering the medium pH from 7.7 to 3.0 and, thus, dissolving the RP. The presence of soil aliquots in the medium controlled the effectiveness of the fungus to increase the concentration of the soluble P. In the presence of soils with a low or medium P sorption capacity, the concentration of the soluble P was high (63.8-146.6 mg L-1) in comparison with the inoculated (soilless) treatment (50.0 mg L-1) and the uninoculated control (0.7 mg L-1). By contrast, with very-high P fixing soil aliquots, the concentration of the soluble P was very low (3.6-33.1 mg L-1); in addition, in these soils, the fungus immobilized more P into its mycelia than in soils with a low or medium P fixation capacity. The capacity of a soil to fix P seems to be a good predictor for the effectiveness of this fungus to increase the soluble P concentration via RP dissolution.