Enumeration Of Spores Research Articles

An assessment of twenty-three mycorrhizal inoculants reveals limited viability of AM fungi, pathogen contamination, and negative microbial effect on crop growth for commercial products

Ensuring sustainable agriculture is crucial amidst global challenges, demanding effective methods to enhance soil health and nutrient cycling. Microbial inoculants, particularly arbuscular mycorrhizal (AM) fungi, offer promising solutions. However, concerns persist regarding the efficacy and quality control of commercial products. Past work assessing commercial inoculants have not controlled for fertilizers added to individual products when assessing product effects under typical use.This study examines twenty-three mycorrhizal inoculants using conventions of organic production to shed light on differences between laboratory grown fungi, commercial products, and field soil. Employing a comprehensive approach, inoculants were assessed through spore enumeration, root infection potential, and crop growth response.The results uncover significant shortcomings in many commercial products compared to laboratory grown fungi. Key findings include discrepancies of up to 100 % in reported propagule counts versus spore concentrations, insufficient root colonization by commercial inoculants, and contamination by fungal plant pathogens, particularly Olpidium, in products. Moreover, while laboratory grown fungi exhibited superior symbiotic relationships with host plants due to increased colonization abilities and crop benefit, commercial inoculants often failed to deliver significant growth benefits when fertilizers are controlled for.These findings highlight the urgent need for improved standards and practices within the commercial inoculant industry.

Microbiological Biodiversity of Regional Cow, Goat and Ewe Milk Cheeses Produced in Poland and Antibiotic Resistance of Lactic Acid Bacteria Isolated from Them.

(1) Unique sensory values of traditional and regional dairy products made them more and more popular among consumers. Lactic acid bacteria naturally occurring in these products can express antibiotic resistance and be a reservoir of antibiotic resistance genes (ARG) in the environment. The aim of the study was to characterize the microbial diversity of twenty regional cheeses produced from non-pasteurized cow, goat and ewe milk, and investigate the phenotypic and genotypic antibiotic resistance (AR) of lactic acid bacteria isolated from these products. (2) Conventional microbiological methods were applied for the enumeration of lactic acid bacteria (lactobacilli and lactococci) and their isolation, and for the enumeration of Enterococcus, Staphylococcus, Enterobacteriaceae and spores. The disc diffusion method was applied for phenotypic AR. The PCR-based methods were used for strain identification, microbiological diversity of cheeses (PCR-DGGE), and for AR gene detection. (3) Among 79 LAB isolates the most frequent species were L. plantarum (n = 18), Leuc. lactis (n = 17), Lc. lactis (n = 11), Leuc. mesenteroides (n = 9) and L. pentosus (n = 8). Additionally, by using the PCR-DGGE method, DNA of L. casei was found in nine products. Lactobacilli (5.63-8.46 log cfu/g) and lactococci (6.15-8.41 log cfu/g) predominated over Enterococcus (max. 4.89 log cfu/g), Staphylococcus (max. 4.18 log cfu/g), and Enterobacteriaceae (mostly up to 4.88 log cfu/g). Analysis of phenotypic resistance to tetracycline (30 µg), erythromycin (15 µg), and chloramphenicol (30 µg) showed that 29% of LAB isolates were resistant to one antibiotic, 8%-to two, and 12%-to all tested antibiotics. Antibiotic resistance genes (AGR) for tetracycline (tet(M), tet(L), tet(W)), erythromycin (erm(B)) and chloramphenicol (cat-TC) were detected in 30 (38%), 29 (36.7%) and 33 (43.4%) LAB isolates, respectively. Among 31 LAB isolates phenotypically susceptible to all tested antibiotics, only 5 (16%) had no ARGs. (4) The results obtained in our work shed light on the potential threat posed by the widespread presence of ARGs in LAB present in regional cheeses.

English

Arbuscular mycorrhizal fungi (AMF) are organisms that can establish symbioses with 80% of terrestrial plants. Soils were collected in three agroecological zones (AEZ) of the Central African Republic (CAR). The aim of this research is to study the diversity of AMF in the CAR. The spore extraction and enumeration, granulometric and physicochemical analyses of each soil were performed. The results showed that AEZ3 has a large number of spores; it is therefore the densest followed by AEZ2 and AEZ1. The particle size analysis showed that AEZ1 and 2 have a sandy-clay texture and AEZ3 is sandy-silty. The average water pH of the three surveyed areas is 5.13. The organic matter content is low. The total nitrogen content of the soils varies from one area to another. The C/N ratio of the studied soils varies from 9.65 to 15.5 with an average of 13.8. The total phosphorus content is lower than the norm. The best calcium content is obtained in AEZ3 followed by AEZ1. Magnesium, sodium, the sum of exchangeable cations and cation exchange capacity (CEC) were below the critical thresholds.   Key words: Diversity, Arbuscular mycorrhizal fungi (AMF), agroecological zones, Central African Republic.

Bacillus spore enumeration using flow cytometry: A proof of concept for probiotic application

Use of flow cytometry (FCM) for bacteria quantification is growing in the food industry. We report here a FCM method using a double-staining LDS751/SYTO24 for the quantification of probiotic Bacillus viable cells and its spores, with potential application for the control of commercial product specifications.

Application of Enzyme Indicators in Hydrogen Peroxide Biodecontamination Cycle Development: A Critical Evaluation of Indicator Variability and Correlation to Biological Indicator Results.

An important prerequisite for aseptic production of pharmaceuticals is the appropriate reduction of microbiological contamination. Hydrogen peroxide is commonly applied as decontamination agent for such processes in barrier systems. The development and validation of the barrier decontamination cycles is usually performed with the use of biological indicators (Geobacillus stearothermophilus). However, these indicators inherit certain drawbacks. A new type of indicators became available lately, based on the use of enzyme as a substance decomposed by hydrogen peroxide. There is no broad experience on the practical application of these enzyme indicators yet. Therefore, we investigated the practical use of enzyme indicators in comparison with biological indicators. To broaden the understanding on how these indicators might be used in practice, we provided an evaluation of indicator response variability and the correlation of enzyme indicator results to biological indicator results. In this study, we found the variability of the quantitative enzyme indicator reading to be < 30%, which is much smaller than the variability for spore enumeration of biological indicators (-50% to +300%). Further, we introduced a universal normalization approach to enable convenient comparison and evaluation of different enzyme indicator results. Finally, we discussed a new approach to establish a quantitative correlation between enzyme indicators and biological indicators with the use of threshold values to extrapolate theoretical biological indicator group results. We take the findings of this study as a basis for a suggestion on the practical application of enzyme indicators. We expect that the discussed possibilities of correlating enzyme indicator results to biological indicator results will allow the combined practical use of both indicator types. This would enable the continued application of proven approaches in new ways. Beyond this, the fast reading of enzyme indicators with the gain of significant quantitative information might result in faster and safer qualification and validation of pharmaceutical production equipment in the future.

A standard set of testing methods reliably enumerates spores across commercial milk powders

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.

Enumeration and Identification of Bacterial Spores in Cocoa Powders

The presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genera Bacillus and Geobacillus are typically present. Spores are a concern in heat-treated foods when they survive heat treatments and the finished product supports germination, growth, and potentially toxin production. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS, respectively) were evaluated, leading to the recommendation of one global method specifically for cocoa powders. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. The method includes dilution of cocoa powder using buffered peptone water, heating at 80°C for 10 min for TMS and TTS counts, and heating at 100°C for 30 min for a heat-resistant (HR) spore count. Tryptic soy agar is used as a recovery medium with a maximal concentration of cocoa powder of 2.5 mg/mL (to prevent growth inhibition) and a nonnutrient agar overlay to prevent swarming of bacteria. Plates are incubated for at least 72 h at 30°C for recovery of mesophilic bacteria and 55°C for thermophilic bacteria. Suitable alternatives to specific method parameters are provided. Median values of total spore concentrations are low (<400 CFU/g for TMS and <75 CFU/g for TTS), and concentrations of HR spores are very low (<5 CFU/g). Importantly, the relation between concentrations of HR spores in cocoa powder and incidence of spoilage of heat-treated beverages containing cocoa is currently unclear. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore-forming species identified (49 and 39%, respectively). Both species are known for high variability in spore heat resistance. The development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders.

Development of fungal spore staining methods for flow cytometric quantification and their application in chlorine-based disinfection.

Fungal contamination in drinking water has been becoming a hot topic. The routine enumeration method of fungal spores is heterotrophic plate counts (HPC). However, this method is time-consuming and labor-intensive and there is also the difficulty of enumerating viable but non-culturable cells. In this study, a rapid, simple and accurate method for quantifying fungal spores and discriminating their viability in water was established using flow cytometry (FCM) combined with fluorescence dyes. The optimal staining conditions are as follows: spores suspensions are sonicated at 495W for 5min as pretreatment, and then 10μL of SYBR Green I (100×) and 30mM Ethylene diamine tetraacetic acid are added to a 500μL water sample, which incubate at 35°C for 20min in dark. The concentration of fungal spores measured by FCM was highly correlated with HPC results and microscope observations, with correlation coefficient of 0.996 and 0.988, respectively. This staining method can be widely applied to the enumeration and viability evaluation of fungal spores. In addition, chlorine-based inactivation of three genera of fungal spores was assessed by plating and FCM. The result showed that all three genera of fungal spores lost culturability firstly and then membrane integrity decreased, preliminarily revealing the inactivation mechanism. The inactivation rate constants of membrane damage varied in the following order: chlorine dioxide>chlorine>chloramine. This study concluded that FCM is an appropriate and alternative tool to detect fungal spores' viability and can be used for evaluating the fungal inactivation by disinfectants.

Occurrence and identification of spore-forming bacteria in skim-milk powders

The different customer and regulatory specifications for mesophilic and thermophilic aerobic and anaerobic spore numbers in skim-milk powder, in addition to some specifications on specific spore-forming bacteria, such as Bacillus cereus, can be challenging for the industry to meet. Twenty-two samples of medium-heat skim-milk spray-dried powder from eight sources were analysed in triplicate with 16 bacterial and spore enumeration tests to understand the variety of spore-forming bacteria population. Using 16S rDNA sequencing, the species were identified for 269 isolates that were representative of the various tests. Of the isolates identified, 68% were Bacillus licheniformis, a facultative anaerobe that can survive and grow at mesophilic and thermophilic temperatures, making it difficult to eliminate in manufacturing environments. Using whole genome sequencing, 16 of 23 isolates identified as B. licheniformis by 16S sequencing were confirmed as B. licheniformis, four were identified as Bacillus paralicheniformis and three were identified as Bacillus sp. H15-1.

Impact of Strain, pH and Ethanol Concentration on Ethanol Activation Method of Bacillus Spores Using a Single Spore Approach

Aims: This work aims to determine the ideal conditions for ethanol activation of spores during their enumeration and compare to thermal activation which is the reference method.&#x0D; Place and duration: Department of Microbiology of the University of Yaoundé I between May2016 and June 2018.&#x0D; Methodology: Bacillus cereus and Bacillus subtilis spores were activated according to an experimental central composite design with three-factor. The factors considered were exposure time, ethanol concentration, pH and activation temperature according to the types of activation. Germination yield was carried out by individually monitoring each spore of a population on solid medium in order to determine the population germination kinetic parameters (time and rate of colony appearance within a population during germination) and germination yields. These parameters were compared with those obtained after thermal activation known as a reference method.&#x0D; Results: The factors strain and pH, significantly influenced the rate of spore germination within the population after ethanol activation. In the case of thermal activation, the specie and activation temperature were the most influential factors. The best germination yields were obtained for alcoholic activation of spores at 30% ethanol for 60min exposure at pH7, while for thermal activation the best yields varied from one strain to another depending on the activation conditions.&#x0D; Conclusion: Ethanol activation can be considered as a good substitute of thermal activation during spore enumeration provided activation conditions are well controlled. This is in our opinion the first detailed study comparing ethanol activation to heat activation of Bacillus spores. It will impact future revisions of spore enumeration protocols proposed by norms that take into consideration spore activation and reduce bias in spore enumeration.

Observations on research with spores of Bacillales and Clostridiales species.

The purpose of this article is to highlight some areas of research with spores of bacteria of Firmicute species in which the methodology too commonly used is not optimal and generates misleading results. As a consequence, conclusions drawn from data obtained are often flawed or not appropriate. Topics covered in the article include the following: (i) the importance of using well-purified bacterial spores in studies on spore resistance, composition, killing, disinfection and germination; (ii) methods for obtaining good purification of spores of various species; (iii) appropriate experimental approaches to determine mechanisms of spore resistance and spore killing by a variety of agents, as well as known mechanisms of spore resistance and killing; (iv) common errors made in drawing conclusions about spore killing by various agents, including failure to neutralize chemical agents before plating for viable spore enumeration, and equating correlations between changes in spore properties accompanying spore killing with causation. It is hoped that a consideration of these topics will improve the quality of spore research going forward.

Assessment of arbuscular mycorrhizal fungal spore density and viability in soil stockpiles of South African opencast coal mines

The symbioses between arbuscular mycorrhizal (AM) fungi and plant roots is essential for nutrient uptake and growth of most vascular plants. Soil condition and management influence the density and viability of arbuscular mycorrhizal fungal (AMF) spores. In this study, AMF spore density and viability in soil stockpiles obtained from three opencast coal mines were assessed. Soil samples were randomly collected from stockpiles and an unmined site at depths of ≤20 cm (topsoil) and >20 cm (subsoil) for enumeration of AMF spores and a mycorrhizal trap culture experiment using maize (Zea mays). Roots of trap-plants were assessed for mycorrhization by classical staining as well as detection of nuclear rRNA gene of the Glomeromycota phylum. Number of AMF spores was highest in topsoil from the unmined site and was significantly (p < 0.05) influenced by site × sampling depth interaction. DNA-based detection revealed that AMF colonisation was mostly associated with topsoil than with subsoil. Five operational taxonomic units (OTUs)/ species related to Paraglomus were identified; however, some of the OTUs were phylogenetically distant from defined Paraglomus species on the basis of sequences in the GenBank database. Overall, results suggest that AMF spore density in stockpiles do not differ from an unmined site, but viability may be influenced by soil depth.

Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and impact of culturing conditions

In this study, different methods were evaluated for enumeration of spores of G. stearothermophilus, different sporulation methods were assessed for yields and wet heat resistances of obtained spores, and subsequently, the variation in heat resistances of spores was determined. Overall, tryptone soya agar (TSA) was the most suitable medium for enumeration of spores of this thermophilic bacterium. Sporulation on different media both at 55 and at 61 °C led to considerable variation in spore heat resistance. The heat resistance of spores was highest upon sporulation on medium supplemented with free ions of calcium, potassium, magnesium and manganese (CaKMgMn). For 18 different G. stearothermophilus strains that were isolated from various sources, spores were subsequently produced on nutrient agar supplemented with CaKMgMn at 55 °C. Strain ATCC 12980T, also known as 9A20, which is commonly used in steam sterilization tests was included. The survival of spores of all strains was assessed at 125 °C and 130 °C using two independent spore batches per strain. The mean D125°C for spores of the 18 strains was 1.1 min (95% PI 0.48–2.3 min) and the mean D130°C was 0.37 min (95% PI 0.17–0.82 min). For spore inactivation of these 18 strains, a z-value of 11.1 °C was estimated, resulting in an estimated D-value of 2.4 min (95% PI 1.1–5.2) at the reference temperature 121.1 °C. Based on the data sets obtained in this study, it was found that the variability in spore heat resistance could largely be attributed to strain variability and conditions used during sporulation (especially the sporulation medium); reproduction and experimental variabilities were much smaller. The established variabilities were compared with the overall variability in spore heat resistance of G. stearothermophilus based on a meta-analysis of reported D-values. The data presented indicate that strain variability and history of sporulation each account for approximately half of the overall variability observed with respect to the heat resistance of spores of G. stearothermophilus. The findings presented in this study allow for optimal recovery of G. stearothermophilus spores from foods and a better understanding of factors that determine the heat resistance properties of spores of G. stearothermophilus. Moreover, this study once more underlines the limited effects of heat treatments used in the food industry on inactivation of spores of this bacterium.

Microbiological Evaluation of Carcasses of Wild Boar Hunted in a Hill Area of Northern Italy

This study evaluated the prevalence of potential pathogenic bacteria (mainly Campylobacter spp., but also Listeria monocytogenes and Salmonella) in wild boar (S us scrofa) and the hygiene of carcasses of wild boar hunted in a hill area of northern Italy during a hunting season (October to December). In total, 62 animals were submitted to microbiological analyses of the tonsils (detection of Listeria spp. and Listeria monocytogenes), caecal content (detection of Salmonella and Campylobacter spp.), mesenteric lymph glands (detection of Salmonella), and carcasses. In addition to analyzing pathogen prevalence and carcass hygiene of these animals, we performed an enumeration of total viable count (TVC), Enterobacteriaceae, Escherichia coli, coagulase-positive staphylococci, and spores of sulfite-reducing clostridia. Influencing factors considered were sex, weight, and age of the animals and environmental temperature on the day of hunting. A high prevalence was observed for L. monocytogenes in tonsils (35.3%) and for Campylobacter spp. in caecal content (51.8%), whereas Salmonella enterica strains (mainly serovar Thompson) were only occasionally isolated (7% in caecal content and 3.5% in lymph glands). The prevalence of L. monocytogenes was influenced by animal age and environmental temperature. Campylobacter spp. were the only pathogens detected on the carcasses (16.7%). Carcasses were characterized by low levels of contamination: TVC, 3.21 ± 0.80 log CFU/cm2, Enterobacteriaceae, 1.32 ± 0.89 log CFU/cm2; E. coli, 1.31 ± 0.93 log CFU/cm2; and occasional detection of low counts of staphylococci and clostridia. TVC was positively influenced only by high environmental temperature, and higher Enterobacteriaceae counts were detected on heavy male carcasses than on females. The results confirmed the potential role of wild boars as reservoirs for the most important foodborne pathogens. But a low carcass contamination level is achievable if hunters are properly trained about hygienic carcass management and slaughtering procedures.

Evaluation of micro-well collector for capture and analysis of aerosolized Bacillus subtilis spores.

Bioaerosol sampling and identification are vital for the assessment and control of airborne pathogens, allergens, and toxins. In-situ analysis of chemical and biological particulate matter can significantly reduce the costs associated with sample preservation, transport, and analysis. The analysis of conventional filters is challenging, due to dilute samples in large collection regions. A low-cost cartridge for collection and analysis of aerosols is developed for use in epidemiological studies and personal exposure assessments. The cartridge collects aerosol samples in a micro-well which reduces particles losses due to the bounce and does not require any coating. The confined particle collection area (dwell~1.4 mm) allows reducing the elution volume for subsequent analysis. The performance of the cartridge is validated in laboratory studies using aerosolized bacterial spores (Bacillus subtilis). Colony forming unit analysis is used for bacterial spore enumeration. Cartridge collection efficiency is evaluated by comparison with the reference filters and found to be consistent with tested flow rates. Sample recovery for the pipette elution is ~80%. Due to the high density of the collected sample, the cartridge is compatible with in-situ spectroscopic analysis and sample elution into the 10–20 μl liquid volume providing a significant increase in sample concentration for subsequent analysis.

Exclosures restored the density and root colonization of arbuscular mycorrhizal fungi in Tigray, Northern Ethiopia

IntroductionExclusion of grazing animals and tree plantations were among the methods used for the restoration of degraded lands in tropical semiarid areas. Exclosures can foster secondary forest succession by improving soil conditions and modifying microclimate for understory growth. This paper compared the arbuscular mycorrhizal fungi (AMF) spore density, root colonization of woody plants and soil chemical properties under exclosure with increasing age, and grazing land at different slope positions.MethodsThe study was conducted in northern Ethiopia from 12 exclosure sites paired each with adjacent grazing land in total from 24 sites with four treatments replicated three times. In the entire study, 216 plots were examined of which 108 were in exclosures and 108 in communal grazing lands. There were four age classes and three slope positions in each of the land uses. Composite soil and root samples were collected using nested plots measuring 100m2 from four sides of 1763 plants for spore enumeration and root colonization. Soils for chemical properties were collected from the four corners and center of 5 m × 5 m plots.ResultsAll the 61 woody plant species that belong to 41 families were colonized by AMF. Spore density and root colonization were significantly higher in exclosures as compared to grazing land and increased with increasing ages of exclosures. Foot slope had significantly higher spore density and root colonization than middle and upper slopes. Soil chemical properties were significantly higher in the exclosure, oldest age of exclosure, and foot slope position (except P). AMF spore density and root colonization were significantly positively correlated with soil chemical properties.ConclusionsExclosures are helpful to restore the AMF spore density and root colonization of woody species and soil fertility.

Re-aerosolization of Bacillus thuringiensis spores from concrete and turf.

Following the malicious and/or accidental release of an aerosol of Bacillus anthracis spores, the immediate risk of human inhalation would decrease as the spores were deposited on surfaces or diluted by wind flow. There is, however, a concern that the deposited spores could become re-aerosolized and so present an ongoing hazard. Using an accurate simulant for B.anthracis spores a method is reported here that allowed the enumeration of re-aerosolized spores from concrete and turf by wind flow and footfall. Under the conditions used, the rates of re-aerosolization were low. These findings will need to be verified under real outdoor conditions before the true significance in terms of secondary exposure to pathogenic spores can be assessed.

Characterizing and handling different kinds of AM fungal spores in the rhizosphere.

Spores are important propagules as well as the most reliable species-distinguishing traits of arbuscular mycorrhizal (AM) fungi. During surveys of AM fungal communities, spore enumeration and spore identification are frequently conducted, but generally little attention is given to the age and viability of the spores. In this study, AM fungal spores in the rhizosphere were characterized as live or dead by vital staining and by performing a germination assay. A considerable proportion of the spores in the rhizosphere were dead despite their intact appearance. Furthermore, morphological and molecular analyses of spores to determine species identity revealed that both viable spores and dead spores with contents were identified. The accurate identification of spores at different developmental stages on the basis of morphology requires considerable experience. Our findings suggest that surveys of AM fungal communities based on spore enumeration and morphological and molecular identification are likely to be inaccurate, primarily because of the large proportion of dead spores in the rhizosphere. A viability check is recommended prior to spore molecular identification, and the use of trap cultures would give more reliable morphological identification results. We show that the abundance and activity of AM fungi in the rhizosphere can be determined by calculating the density of viable spores and the density of spores that could germinate. The adoption of these methods should provide a more reliable basis for further AM fungal community analysis.

Spore test parameters matter: Mesophilic and thermophilic spore counts detected in raw milk and dairy powders differ significantly by test method

United States dairy industry exports have steadily risen in importance over the last 10yr, with dairy powders playing a particularly critical role. Currently, approximately half of US-produced nonfat dry milk and skim milk powder is exported. Reaching new and expanding existing export markets relies in part on the control of endospore-forming bacteria in dairy powders. This study reports baseline mesophilic and thermophilic spore counts and spore populations from 55 raw material samples (primarily raw milk) and 33 dairy powder samples from dairy powder processors across the United States. Samples were evaluated using various spore testing methodologies and included initial heat treatments of (1) 80°C for 12 min; (2) 100°C for 30 min; and (3) 106°C for 30 min. Results indicate that significant differences in both the level and population of spores were found for both raw milk and dairy powders with the various testing methods. Additionally, on average, spore counts were not found to increase significantly from the beginning to the end of dairy powder processing, most likely related to the absence of biofilm formation by processing plant-associated sporeformers (e.g., Anoxybacillus sp.) in the facilities sampled. Finally, in agreement with other studies, Bacillus licheniformis was found to be the most prevalent sporeformer in both raw materials and dairy powders, highlighting the importance of this organism in developing strategies for control and reduction of spore counts in dairy powders. Overall, this study emphasizes the need for standardization of spore enumeration methodologies in the dairy powder industry.

A polymerase chain reaction assay for ascosporic inoculum of Sclerotinia species

A polymerase chain reaction (PCR) assay was developed that amplified a 170-bp fragment of the intergenic spacer region of Sclerotinia sclerotiorum, the cause of white mould. Sensitivity was 10 S. sclerotiorum ascospores per DNA extraction (0.2 ascospores per PCR reaction). The presence of soil did not affect sensitivity at 50, 100 and 500 ascospores/DNA extraction, but reduced sensitivity at 25 and 10 ascospores/DNA extraction by 10% and 30%, respectively. The assay did not amplify DNA of Botrytis cinerea but detected S. minor and S. trifoliorum. Utility of the test for detection of S. sclerotiorum ascospores in bean fields was demonstrated using rotating impaction samplers over two seasons. The use of the test in combination with an impaction sampler may provide benefits in time, sensitivity and specificity compared with visual identification and enumeration of spores from traps only. This system may provide an opportunity to schedule fungicides during periods of inoculum presence for disease management.