Test method development to evaluate hot,humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores

Aims

To develop test methods and evaluate the survival of Bacillus anthracis ?Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air.

Methods and Results

Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH‐adjusted bleach decontamination.

Conclusions

Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ?Sterne and B. thuringiensis Al Hakam spores with similar kinetics.

Significance and Impact of the Study

Hot, humid air is a potential alternative to conventional chemical decontamination.     

Adaptation of the Spore Discharge Mechanism in the Basidiomycota

Background

Spore discharge in the majority of the 30,000 described species of Basidiomycota is powered by the rapid motion of a fluid droplet, called Buller''s drop, over the spore surface. In basidiomycete yeasts, and phytopathogenic rusts and smuts, spores are discharged directly into the airflow around the fungal colony. Maximum discharge distances of 1–2 mm have been reported for these fungi. In mushroom-forming species, however, spores are propelled over much shorter ranges. In gilled mushrooms, for example, discharge distances of <0.1 mm ensure that spores do not collide with opposing gill surfaces. The way in which the range of the mechanism is controlled has not been studied previously.

Methodology/Principal Findings

In this study, we report high-speed video analysis of spore discharge in selected basidiomycetes ranging from yeasts to wood-decay fungi with poroid fruiting bodies. Analysis of these video data and mathematical modeling show that discharge distance is determined by both spore size and the size of the Buller''s drop. Furthermore, because the size of Buller''s drop is controlled by spore shape, these experiments suggest that seemingly minor changes in spore morphology exert major effects upon discharge distance.

Conclusions/Significance

This biomechanical analysis of spore discharge mechanisms in mushroom-forming fungi and their relatives is the first of its kind and provides a novel view of the incredible variety of spore morphology that has been catalogued by traditional taxonomists for more than 200 years. Rather than representing non-selected variations in micromorphology, the new experiments show that changes in spore architecture have adaptive significance because they control the distance that the spores are shot through air. For this reason, evolutionary modifications to fruiting body architecture, including changes in gill separation and tube diameter in mushrooms, must be tightly linked to alterations in spore morphology.     

Glomeromycotan mycorrhizal fungi from tropical Australia III. Measuring diversity in natural and disturbed habitats

Aims and Background

The aim was to investigate the diversity and distribution of Glomeromycotan fungi forming arbuscular mycorrhizal associations (AMF) in undisturbed and disturbed habitats in the vicinity of Kakadu National Park in tropical Australia. This is a tropical region with a 7–9 month dry season and a monsoonal wet season. Complimentary methods of fungus detection were used to investigate the diversity and relative dominance of AMF at a regional scale.

Methods

Soils were sampled from 32 sites, representing eucalypt savanna woodlands, wetlands, sandstone escarpment, rainforest, and disturbed mine waste rock dumps (overburden or spoil). Populations of AMF were identified and quantified using spores from soil. Morphology patterns of fungi colonising bait plant roots were examined and isolates were obtained by four complimentary pot-culturing methods.

Results

Different methods of detecting fungi produced different answers about which AMF were most important in the tested soils. In particular, spore surveys apparently underestimated the importance of Glomus species and overestimated the activity of Acaulospora species with numerous small spores, while calculated spore biovolumes overestimated the importance of Scutellospora and Gigaspora species with large spores, relative to inoculum levels of these fungus categories measured in bioassays. Spore surveys revealed 15 species of fungi and 8 additional fungi were recovered from the same soil samples using pot-culture isolation methods. Pot-cultures were especially important for detecting Glomus species that had high inoculum levels, but rarely produced spores in soils. Spores of AMF increased in abundance as vegetation developed in mine habitats reaching a peak that was higher than in undisturbed plant communities. Spore numbers (but not biovolumes) were well correlated with bioassay measurements of inoculum levels.

Conclusions

Most AMF species were widespread, but several were restricted to disturbed habitats or wetland soils. Undisturbed sites had a substantially higher diversity of AMF than partially vegetated mine waste rock dumps. It is recommended that AMF population surveys should not be based entirely on spore occurrence data, to avoid overlooking important fungi that sporulate infrequently. These fungi could be detected by bioassays or pot culture isolation from soil. Major variations in the detectability of AMF correspond to different life history strategies and can mask variations in their abundance.     

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco

Key message

Phylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen.

Abstract

Tobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene—as a fusion with the GFP gene—targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.     

Building filaments in the air: aerial morphogenesis in bacteria and fungi

To disperse their spores to new sites, filamentous fungi and bacteria need to erect aerial filaments, which develop into fruiting bodies and spore-bearing structures. The first challenge to aerial development is breaking surface tension at an aqueous-air interface, and in both groups of microorganisms, surface-active proteins take part in the initiation of aerial morphogenesis. Comparative analysis of fungi and bacteria is providing new insights into the means by which aerial filamentation is accomplished.     

Morphological characterization and germination of aerial and submerged spores of the entomopathogenic fungus Verticillium lecanii

Under solid-state and liquid-state cultivations, the entomopathogenic fungus Verticillium lecanii F091 produced different types of spores. The aerial spores (AS) on cooked rice formed clusters on the tips of conidiophores, while the submerged spores (SS) were dispersed in the medium. The aerial spore appeared relatively uniform in size, which was 6.1 ± 0.9 m long, and 2.2 ± 0.3 m wide. The submerged spore varied in shape and size, with a mean length of 5.0 ± 1.0 m and width of 1.9 ± 0.5 m. Under scanning electron microscopy, the AS had a tendency to have rough, brittle surface characteristics; however, the SS appeared smooth on the surface. These spores were compared in two different germination media. On SMAY (Sabouraud maltose, agar, yeast extract, and neopeptone) coated coverslips, the AS did not show germ tubes until 8 h of incubation; while the SS showed many germ tubes. However, over 90% spore germination ratio was reached for both types of spores at 18-h of incubation. In the liquid medium, the SS germinated rapidly and many spores even produced spores on the spores; while the AS germinated, grew, and branched in the submerged culture gradually, and some sporulated on the tips of the short branches, or on the mycelia until 18 h of incubation. Evidently, the germination, growth patterns of aerial or submerged spores differed greatly under the different culture conditions. The virulence of the pathogen in relation to the type of spore of V. lecanii is discussed.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Structural Proteins Involved in Emergence of Microbial Aerial Hyphae

Filamentous fungi and filamentous bacteria (i.e., the streptomycetes) belong to different kingdoms that diverged early in evolution. Yet, they adopted similar lifestyles. After a submerged feeding mycelium has been established, hyphae grow into the air and form aerial structures from which (a)sexual spores can develop. These spores are dispersed and can give rise to a new mycelium. Some of the key processes involved in the formation of aerial hyphae by these microbes appear to be very similar. In both cases molecules that lower the surface tension are secreted into the aqueous environment, thereby enabling hyphae to grow into the air. Aerial hyphae are then covered with a hydrophobic film. In fungi, this film is characterized by a mosaic of parallel rodlets, while similar rodlets have also been observed on aerial structures of filamentous bacteria. Although the erection of aerial hyphae in both filamentous fungi and filamentous bacteria is dependent upon (poly)peptides that are structurally unrelated, they can, at least partially, functionally substitute for each other.     

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

Background

Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores.

Results

Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP₃) levels and the effect of added IP₃ uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP₅ phosphatase leading to high IP₃ levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP₃ levels; addition of exogenous IP₃ to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP₃ levels remain high, also at unfavourable environmental conditions.

Conclusions

The results imply that environmental conditions regulate PLC activity and that IP₃ induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP₃-forming pathway.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Background  

In order to initiate plant infection, fungal spores must germinate and penetrate into the host plant. Many fungal species differentiate specialized infection structures called appressoria on the host surface, which are essential for successful pathogenic development. In the model plant pathogen Magnaporthe grisea completion of mitosis and autophagy cell death of the spore are necessary for appressoria-mediated plant infection; blocking of mitosis prevents appressoria formation, and prevention of autophagy cell death results in non-functional appressoria.     

Airborne fungi associated with ornamental plant propagation in greenhouses

The objective was to determine potential exposure to airborne fungi in greenhouses and to characterize the temporal patterns of airborne fungi in relation to environmental conditions. We analyzed air samples collected in two greenhouses. Results showed that the top 5 fungi in greenhouse 1 were Trichoderma, hyphal fragments, Aspergillus/Penicillium-like, Cladosporium, and Botrytis in a descending order. Those in greenhouse 2 were Aspergillus/Penicillium-like, Cladosporium, Botrytis, yeast-like, and hyphal fragments. Maximum concentrations of Trichoderma and total spores in greenhouse 1 were 36,426 and 49,729 spores/m³, respectively. Maximum concentrations of Aspergillus/Penicillium and total spores in greenhouse 2 were 46,961 and 71,037 spores/m³, respectively. Airborne fungal populations fluctuated dramatically within 2 h during work hours, tenfold for Aspergillus/Penicillium, 66-fold for Trichoderma, and sevenfold for total spores. QPCR detected Trichoderma harzianum ranging from 7 to 3,500 conidia E/m³. Aspergillus/Penicillium and Botrytis showed diurnal patterns, but not Trichoderma. Aspergillus/Penicillium and Cladosporium were positively correlated with temperature, relative humidity, dew point, heat index, and light and negatively with air movement and air pressure. Botrytis and Trichoderma were not correlated with the environmental factors. Greenhouse workers were potentially exposed up to 71,037 spores/m³ of airborne fungi.     

Detection and identification by PCR of <Emphasis Type="Italic">Clostridium chauvoei</Emphasis> in clinical isolates,bovine faeces and substrates from biogas plant

Background  

Clostridium chauvoei causes blackleg, an acute disease associated with high mortality in ruminants. The apparent primary port of entry is oral, during grazing on pasture contaminated by spores. Cases of blackleg can occur year after year on contaminated pastures. A method to determine the prevalence of C. chauvoei spores on pasture would be useful.     

Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact‐cell MALDI‐TOF mass spectrometry (ICMS)

Aims

To characterize fungal antagonistic bacilli isolated from aerial roots of banyan tree and identify the metabolites responsible for their antifungal activity.

Methods and Results

Seven gram positive, endospore‐forming, rod‐shaped endophytic bacterial strains exhibiting a broad‐spectrum antifungal activity were isolated from the surface‐sterilized aerial roots of banyan tree. The isolates designated as K1, A2, A4 and A12 were identified as Bacillus subtilis, whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using Biolog Microbial Identification System. The antifungal lipopeptides, surfactins, iturins and fengycins with masses varying in the range from m/z 900 to m/z 1550 could be detected using intact‐cell MALDI‐TOF mass spectrometry (ICMS). On the basis of mass spectral and carbon source utilization profile, all seven endophytes could be distinguished from each other. Furthermore, ICMS analysis revealed higher extent of heterogeneity among iturins and fengycins produced by B. subtilis K1, correlating well with its higher antifungal activity in comparison with other isolates.

Conclusion

Seven fungal antagonistic bacilli were isolated from aerial roots of banyan tree, exhibiting broad spectrum of antifungal activity, among which B. subtilis K1 isolate was found to be most potent. The ICMS analysis revealed that all these isolates produced cyclic lipopeptides belonging to surfactin, iturin and fengycin families and exhibited varying degree of heterogeneity.

Significance and Impact of the study

The endophytes are considered as a potential source of novel bioactive metabolites, and this study describes the potent fungal antagonistic bacilli from aerial roots of banyan tree. The isolates described in this study have a prospective application as biocontrol agents. Also ICMS analysis described in this study for characterization of antifungal metabolites produced by banyan endophytic bacilli may be used as a high throughput tool for screening of microbes producing novel cyclic lipopeptides.     

Interactive influence of light intensity and soil fertility on root-associated arbuscular mycorrhizal fungi

Background and aims

Soil nutrients and light have major effects on the economics of arbuscular mycorrhizal (AM) symbioses. This study tests the main and interactive effects of soil fertility and light on AM fungal community.

Methods

We conducted a 3 year mesocosm experiment with a full two factorial design: light (full light or shade) and soil fertility (unfertilized or fertilized), on the Qinghai-Tibetan Plateau. Plant traits, soil characteristics and the AM fungal communities inside roots and in soils were measured.

Results

Shade reduced AM colonization of roots, fertilization reduced the hyphal abundance in the soil, and both factors reduced species richness of AM fungi inside plant roots. Fertilization exacerbated the negative impacts of shade on AM fungal abundance and diversity. We observed 15 phylotypes of AM fungi inside roots and ten morphotypes of AM fungal spores in the soil. Taxa responded differently to shade and fertilization and there was little congruence between the responses of fungi inside the roots and in the spore community.

Conclusions

Our findings indicate that both shade and fertilization reduce the abundance of AM fungi, but the two factors have different effects on the quality of plant roots as habitat for AM fungi.     

A genetic algorithm-Bayesian network approach for the analysis of metabolomics and spectroscopic data: application to the rapid identification of Bacillus spores and classification of Bacillus species

Background  

The rapid identification of Bacillus spores and bacterial identification are paramount because of their implications in food poisoning, pathogenesis and their use as potential biowarfare agents. Many automated analytical techniques such as Curie-point pyrolysis mass spectrometry (Py-MS) have been used to identify bacterial spores giving use to large amounts of analytical data. This high number of features makes interpretation of the data extremely difficult We analysed Py-MS data from 36 different strains of aerobic endospore-forming bacteria encompassing seven different species. These bacteria were grown axenically on nutrient agar and vegetative biomass and spores were analyzed by Curie-point Py-MS.     

Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of <Emphasis Type="Italic">Gracilaria vermiculophylla </Emphasis>(Ohmi) Papenfuss (Gracilariales,Rhodophyta)

Background  

Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae.     

Evidence for separate translocation pathways in determining cadmium accumulation in grain and aerial plant parts in rice

Background  

Cadmium (Cd) translocation and accumulation in the grain and aerial plant parts of rice (Oryza sativa L.) is an important aspect of food safety and phytoextraction in areas with contaminated soil. Because control of Cd translocation and accumulation is likely to be determined by the plants genetics, the Cd contents of grain and the aerial parts of rice may be manipulated to improve food safety and for phytoextraction ability. This study studied Cd translocation and accumulation and their genetic control in aerial parts of rice to provide a starting point for improving food safety and phytoextraction in Cd-contaminated soils.     

Interaction of the heterotrimeric G protein alpha subunit SSG-1 of <Emphasis Type="Italic">Sporothrix schenckii</Emphasis> with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

Background  

Important biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins.