Low Spore Concentrations Research Articles

Intensified biochip system using chemiluminescence for the detection of Bacillus globigii spores

This paper reports the first intensified biochip system for chemiluminescence detection and the feasibility of using this system for the analysis of biological warfare agents is demonstrated. An enzyme-linked immunosorbent assay targeting Bacillus globigii spores, a surrogate species for Bacillus anthracis, using a chemiluminescent alkaline phosphatase substrate is combined with a compact intensified biochip detection system. The enzymatic amplification was found to be an attractive method for detection of low spore concentrations when combined with the intensified biochip device. This system was capable of detecting approximately 1 × 105Bacillus globigii spores. Moreover, the chemiluminescence method, combined with the self-contained biochip design, allows for a simple, compact system that does not require laser excitation and is readily adaptable to field use.FigureSchematic diagram of the miniature biochip detection system

Comparison of Field, Greenhouse, and Detached-Leaf Evaluations of Soybean Germplasm for Resistance to Phakopsora pachyrhizi.

Fourteen soybean accessions and breeding lines were evaluated for resistance to soybean rust caused by the fungus Phakopsora pachyrhizi. Evaluations were conducted in replicated experiments in growth chambers using detached leaves and under greenhouse and field conditions. In growth-chamber experiments, inoculation of detached leaves with 1 × 106 spores/ml resulted in a significantly (P < 0.0001) higher total number of pustules and spores per unit leaf area than inoculations with lower spore concentrations. Amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color in detached leaves. Leaf pieces on a medium containing kinetin at 10 mg/liter had 5% chlorosis at 18 days after plating compared with leaf pieces on media amended with all other plant hormones, which had higher levels of chlorosis. Leaf age significantly affected number of pustules (P = 0.0146) and number of spores per pustule (P = 0.0088), and 3- to 4-week-old leaves had a higher number of pustules and number of spores per pustule compared with leaves that were either 1 to 2 or 5 to 6 weeks old. In detached-leaf and greenhouse screening, plants were evaluated for days to lesion appearance, days to pustule formation, days to pustule eruption, lesion number, lesion diameter, lesion type, number of pustules, and spores per pustule in 1-cm2 leaf area. Plants also were evaluated for diseased leaf area (in greenhouse and field screening) and sporulation (in field screening) at growth stage R6. There were significant (P < 0.0001) differences among genotypes in their response to P. pachyrhizi infection in the detached-leaf, greenhouse, and field evaluations. Accessions PI 594538A, PI 417089A, and UG-5 had very low levels of disease compared with the susceptible checks and all other genotypes. Detached-leaf, greenhouse, and field results were comparable, and there were significant correlations between detached-leaf and greenhouse (absolute r = 0.79; P < 0.0001) and between detached-leaf and field resistance (absolute r = 0.83; P < 0.0001) across genotypes. The overall results show the utility of detached-leaf assay for screening soybean for rust resistance.

Role ofBacillus anthracis Spore Structures in Macrophage Cytokine Responses

The innate immune response of macrophages (Mphi) to spores, the environmentally acquired form of Bacillus anthracis, is poorly characterized. We therefore examined the early Mphi cytokine response to B. anthracis spores, before germination. Mphi were exposed to bacilli and spores of Sterne strain 34F2 and its congenic nongerminating mutant (DeltagerH), and cytokine expression was measured by real-time PCR and an enzyme-linked immunosorbent assay. The exosporium spore layer was retained (exo+) or removed by sonication (exo-). Spores consistently induced a strong cytokine response, with the exo- spores eliciting a two- to threefold-higher response than exo+ spores. The threshold for interleukin-1beta (IL-1beta) production by wild-type Mphi was significantly lower than that required for tumor necrosis factor alpha expression. Cytokine production was largely dependent on MyD88, suggesting Toll-like receptor involvement; however, the expression of beta interferon in MyD88-/- Mphi suggests involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis spore is not immunologically inert, (ii) the exosporium masks epitopes recognized by the Mphi, (iii) the Mphi cytokine response to B. anthracis involves multiple pattern recognition receptors and signaling pathways, and (iv) compared to other cytokines, IL-1beta is expressed at a lower spore concentration.

Persistence and mobility of a Clostridium botulinum spore population introduced to soil with spiked compost

In a recent study it could be shown that compost samples can contain Clostridium botulinum. It was investigated if C. botulinum introduced with compost into botulinum-free soil can persist and be translocated within the soil. Compost was spiked with two C. botulinum type D spore concentrations (10(3) and 10(5) spores g(-1)) and the composts were spread on an experimental site. Over a period of 939 days, samples were taken from the upper (0-5 cm) and the lower (10-30 cm) soil horizons. Physical and chemical as well as microbiological variables were measured. Clostridium botulinum spores were quantified in a culture MPN-PCR assay. On day 757 the last positive sample was obtained in the plots with the lower spore concentration (10(3) g(-1)). The bacteria were never detected in the samples taken from the lower horizons of these plots. Clostridium botulinum persisted over the whole investigation period in the plots which were treated with compost spiked with 10(5) spores g(-1). The concentrations found were between 20 and 20,000 spores g(-1) soil. The bacteria were vertically translocated and could be found in the lower soil horizons (20-2000 spores g(-1) soil) starting 70 days after the compost was spread.

Bacillus anthracis incident, Kameido, Tokyo, 1993.

In July 1993, a liquid suspension of Bacillus anthracis was aerosolized from the roof of an eight-story building in Kameido, Tokyo, Japan, by the religious group Aum Shinrikyo. During 1999 to 2001, microbiologic tests were conducted on a liquid environmental sample originally collected during the 1993 incident. Nonencapsulated isolates of B. anthracis were cultured from the liquid. Multiple-locus, variable-number tandem repeat analysis found all isolates to be identical to a strain used in Japan to vaccinate animals against anthrax, which was consistent with the Aum Shinrikyo members’ testimony about the strain source. In 1999, a retrospective case-detection survey was conducted to identify potential human anthrax cases associated with the incident, but none were found. The use of an attenuated B. anthracis strain, low spore concentrations, ineffective dispersal, a clogged spray device, and inactivation of the spores by sunlight are all likely contributing factors to the lack of human cases.

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l−1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 108 spores ml−1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 108 spores ml−1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 108 spores ml−1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.

A miniature biochip system for detection of aerosolized Bacillus globigii spores.

The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a compact biochip detection system, which includes a miniature diode laser for excitation. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air. Moreover, the incorporation of the miniature diode laser with the self-contained biochip design allows for a compact system that is readily adaptable to field use. In addition, these studies have included investigations into the tradeoff between assay time and sensitivity.

The immune response of rainbow trout (Oncorhynchus mykiss) againstAphanomyces invadans

Aphanomyces invadans, also referred toA. invaderisorA. piscicida, is the aetiological agent of epizootic ulcerative syndrome (EUS). Immune responses of rainbow trout (Oncorhynchus mykiss) against this fungus were examined, including both antibody and macrophage responses. The fish antibodies to the fungus, assessed by an enzyme-linked immunosorbent assay (ELISA) were generally non-specific, but sera from fish vaccinated withA. invadansreacted with bands at 55 and 40kDa by Western blot analysis. Macrophage activity was examinedin vitroby culturing rainbow trout head kidney macrophages with fungal spores. The macrophages were able to phagocytose sporesin vitroand, in some cases, hyphae were observed growing from germinated spores within the macrophage. When low concentrations of spores (1×102and 1×103spore ml−1) were cultured with macrophage monolayers, no germination of spores or growth of hyphae was observed. At concentrations of 1×104spore ml−1or higher, macrophages were unable to kill all the spores and fungal mycelium eventually overgrew the culture. Transmission electron microscopy (TEM) was used to examine the ultrastructural interaction between macrophages and the fungus. The possible role of rainbow trout as a model forA. invadansinfection in salmonids is also discussed.

Dipicolinic acid (DPA) assay revisited and appraised for spore detection.

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.

Fungal spore concentrations in the atmosphere at the Anatolia quarter of Istanbul, Turkey.

In this research, spore concentrations of Cladosporium, Alternaria, Epicoccum, Botrytis, Leptosphaeria, Polythrincium, ascospores, Aspergillus, Penicillium, basidiospores, uredospores, Ustilago, Torula, Erysiphe, Ganoderma, Hyaline indeterminate and others in the atmosphere have been determined, and comparisons have been made between locations with both low and high spore concentrations. The importance of these air-borne fungal spores which cause allergy and asthma are emphasized in the paper from the viewpoint of human health.

Fungal spores are an important component of library air

The airborne fungal spore types were studied in different libraries in Delhi, using an Andersen sampler and a Burkard personal sampler, for culturable and non-culturable fungi respectively. The concentration inside the libraries, before and after the agitation of books, were compared with outside air. The major contributors to the library air areCladosporium, aspergilli/penicillia, smuts andAlternaria, varying from 50 to 14%. Some fungi (Cladosporium, Alternaria, smut,Penicillium chrysogenum andnigricans) showed seasonal occurrence, corresponding to their occurrence in the extramural environment. Aspergilli/penicillia,Drechslera, Curvularia andAspergillus flavus had a significantly higher concentration (P<0.01) inside the library, and recorded a significant increase in concentration after agitation of books. Air-conditioned libraries have low fungal spore concentrations, as compared to naturally ventilated libraries.

Defence related reactions of seedling roots of Norway spruce to infection by Heterobasidion annosum (Fr.) Bref.

Norway spruce seedlings were cultivated in sterile conditions. Roots were infected with a concentration series of germinating conidiospores of Heterobasidion annosum (101–106 ml−1). In other experiments, roots were treated with either mycelial preparations of H. annosum, other wood inhabiting fungi, with protein fractions of culture filtrates of H. annosum, or with chemical elicitors. Successive steps observed during infection were necrosis, formation of phenolics and increasing lignification of cortex and endodermis, colonization of meristem and finally of the stele. High spore concentrations caused necrosis and invasion within 48–72 h; these processes were delayed at low spore concentrations. Lyophilized culture filtrates of H. annosum caused a greater hypersensitive response than protein fractions but less than NaCl, Polygalacturonic acid or ethephon. Mycelial homogenate from nine other wood inhabiting fungi (saprophytes/parasites) induced a hypersensitive response to various extents but this was not correlated to their degree of cross-reactivity with a polyclonal antibody to H. annosum [enzyme-linked immunosorbent assay (ELISA)]. Peroxidase activity increased (two-threefold) in roots infected with H. annosum and one acidic isozyme was considered responsible for the increase in peroxidase. Using immunohistochemical and enzyme staining, peroxidase was found mainly in the cortical/endodermal regions of roots. Cytochemical labelling using anti-peroxidase and immunogold demonstrated increased peroxidase activity in cell walls, papillae and uninvaded middle lamellar cell corners of infected roots.

Studies on the infection of Norway spruce roots by Heterobasidion annosum

The interaction between the root rot fungus (Heterobasidion annosum) and seedling roots of Norway spruce was studied using scanning and transmission electron microscopy, light microscopy, and immunohistochemistry. Results showed that attachment to root surface and penetration of root epidermal walls were facilitated using pregerminated conidiospores as inocula. High inoculum concentrations (107 spores/mL) were also required for rapid colonization and establishment of the pathogen in the cortex. At lower spore concentrations, fewer hyphae were detected within the cortical region. The induction of hypersensitive response was a function of both time and spore concentration. Formation of infective structures on the root surface was evident 24 h after inoculation. Internal colonization of cortical tissues started 24–48 h postinoculation and reached the endodermis within 72 h. Severe destruction of stelar cells was observed 12 – 15 d postinfection. Cell invasion by the pathogen was correlated with formation of papillae and lignification in cortical cells. Increases in the enzymatic activity of defence-related proteins (chitinase and peroxidase) of infected roots were also evident. When vascular tissues were severely damaged disruption of endodermal cells was also recorded using light microscopy. The presence of H. annosum antigenic materials within cell wall boundaries suggests intercellular penetration by the pathogen. Key words: Norway spruce, Heterobasidion annosum, histochemistry, immunofluorescence, chitinase, peroxidase.

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 μg vs 83 μg, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC 50 of about 600 spores/ml.

Influence of Resveratrol on Germination of Conidia and Mycelial Growth of Botrytis cinerea and Phomopsis viticola

Abstract After inoculation of agar substrates containing resveratorl (an inducible stilbene characteristic for Vitaceae) with low concentrations (500/cm2) of Botrytis spores, developing germ tubes showed malformations and growth stopped. High, density inoculations (10,000/cm2) resulted in apparently normal growth, but stress conditions were indicated by the formation of a large number of sclerotia after 14 days. By contrast, growth of Botrytis and Phomopsis colonies starting from pieces of mycelium was slowed down by resveratrol concentrations 50 ppm and stopped by higher concentrations. More than 500 ppm (suspensions) induced rhythmical growth; at the front of the mycelium a clear zone appeared, which was free fromstilbene crystals but exhibited a brownish pigmentation. In liquid cultures pigmentation but no growth inhibition was observed. It is assumed that resveratrol is oxidized by fungal laccases whose isoenzyme bands can be stained with resveratrol after isoelectric focusing. The different effects of this substrate on fungal growth in liquid and on solid media is explained by the localized action of laccases on solid substances where the radius of diffusion is limited.

Environmental factors in allergic bronchopulmonary aspergillosis.

Many workers have suggested that exposure to an environment rich in Aspergillus spores is aetiologically significant in allergic bronchopulmonary aspergillosis. This hypothesis has been investigated by means of an environmental survey in which eight urban dwelling patients with this disease and five atopic control patients took part. Enquiries were made about contact with known sources of Aspergillus fumigatus and air spora studies were conducted in the home. Findings were similar for both groups. No high spore concentrations were recorded in the homes of either group. Patients with allergic bronchopulmonary aspergillosis had not been more exposed to potentially rich sources of A. fumigatus than the atopic control patients. The view is expressed that specific host susceptibility is more important in the pathogenesis of allergic bronchopulmonary aspergillosis than environmental factors. Ince a patient is sensitized however minor increases in spore concentration can cause symptomatic disease producing the recognized winter exacerbations of allergic bronchopulmonary aspergillosis. Possible mechanisms allowing the development of the disease in the presence of low spore concentrations are discussed.

Fungitoxic and phytotoxic effect of some surface‐active agents applied for the control of apple powdery mildew

SUMMARYMixtures of methyl esters of fatty acids (‘Off‐Shoot O’) and mixtures of fatty alcohols (‘Off‐Shoot T’), applied during the late autumn (November), eradicated apple powdery mildew (caused by Podosphaera leucotricha) from infected buds. Applications in the spring at bud‐burst were less effective.The autumn sprays caused little damage to the cultivars Bramley's Seedling, Cox's Orange Pippin and Golden Delicious but on Worcester Pearmain 50% of the buds were killed by a 5% Off‐Shoot O spray. The bud‐burst sprays were damaging and reduced yield on the Cox and Bramley trees.A 5% a.i. mixture of nonanol/lissapol applied to a range of commercial cultivars was effective in eradicating powdery mildew but was more phytotoxic than the Off‐Shoot compounds. Spraying Cox trees later in the winter failed to reduce the damage.Eradication of the overwintering stage of the disease resulted in low spore concentrations during the following spring and early summer, enabling some economies to be made in the summer mildew fungicide programme.

Localized infection with tobacco necrosis virus protects cucumber against Colletotrichum lagenarium

A cultivar of cucumber susceptible to Colletotrichum lagenarium race 1 was systemically protected against disease caused by the fungus by prior infection with tobacco necrosis virus (TNV). Infection of one cotyledon with the virus protected the opposite cotyledon and the first true leaf against disease caused by a subsequent infection with C. lagenarium. Autoclaved sap from TNV-infected plants or sap from non-inoculated plants did not elicit protection. Infection with the virus prior to infection with C. lagenarium caused a reduction in the number and size of lesions caused by the fungus at low spore concentrations, but was less effective against higher spore concentrations. The number of lesions formed on a TNV-infected cotyledon was not affected by prior infection of the opposite cotyledon with TNV or C. lagenarium.

Sources and incidence of airborne Aspergillus fumigatus (Fres).

Specific surveys of the air for Aspergillus fumigatus were carried out in rural and urban situations over a 2-year period. Overall, low concentrations of spores were recorded with a higher incidence during the "winter" months. Counts in the open air and in a hospital ward showed similar fluctuations, the indoor counts being consistently lower. Plant debris in the form of compost heaps and stacks of hay and straw baled with a high moisture content in which self-heating occurs, produces large numbers of spores which may be liberated into the air causing high but localized counts if disturbed. The widespread distribution of decaying leaves following leaf fall represents a potential source of smaller concentrations of spores but over a much larger area. This availability of decaying plant debris with high water content fulfils the growth requirements of Aspergillus fumigatus and is the probable explanation of its winter seasonality.

Germination studies with Geotrichum candidum

Arthrospores of Geotrichum candidum Link germinate in the absence of exogenous nutrients at low spore concentrations but at high spore concentrations show self-inhibition. Nutrients can overcome the self-inhibition, and there is a relationship between the spore concentration and the nutrient level required to overcome the inhibition. Early in germination the mitochondria in the spore change in form from short and oval to filamentous, and the change precedes an increase in oxygen uptake. Young spores have a short lag phase, older spores a longer one, and this difference is correlated with changes in degree of vacuolation of the spores.