The role of temperature and carbon dioxide climatic stress factors on the growth kinetics of Escherichia coli.

AIMS: The global level of carbon dioxide and temperature in the atmosphere is expected to increase, which may affect the survival of the stress-adapted bacteria. In this study, the effect of temperature and dissolved carbon dioxide on the growth rate of Escherichia coli-eGFP tagged strain was studied, thus assessing its response to induced environmental stress factors. METHODS AND RESULTS: A kinetic assay has been performed using a microplate reader with a spectrofluorometer to determine the specific growth rates. Polynomial models were developed to correlate the environmental conditions of temperature and carbon dioxide with Escherichia coli BL21 (DE3) growth in culture media and dairy by-products. At a temperature of 42°C, as the dissolved CO2 increased, a decrease in µmax by 0.76 h-1 was observed. In contrast, at 27°C, this increase led to an increase in µmax by 0.99 h-1. Moreover, a correction factor was added when applying the model to dairy whey samples. CONCLUSIONS: The application of this developed model can be considered a useful tool for predicting the growth of Escherichia coli using climate projections.

Microbial decontamination assisted by ultrasound-based processing technologies in food and model systems: A review.

Ultrasound (US) technology is recognized as one of the emerging technologies that arise from the current trends for improving nutritional and organoleptic properties while providing food safety. However, when applying the US alone, higher power and longer treatment times than conventional thermal treatments are needed to achieve a comparable level of microbial inactivation. This results in risks, damaging food products' composition, structure, or sensory properties, and can lead to higher processing costs. Therefore, the US has often been investigated in combination with other approaches, like heating at mild temperatures and/or treatments at elevated pressure, use of antimicrobial substances, or other emerging technologies (e.g., high-pressure processing, pulsed electric fields, nonthermal plasma, or microwaves). A combination of US with different approaches has been reported to be less energy and time consuming. This manuscript aims to provide a broad review of the microbial inactivation efficacy of US technology in different food matrices and model systems. In particular, emphasis is given to the US in combination with the two most industrially viable physical processes, that is, heating at mild temperatures and/or treatments at elevated pressure, resulting in techniques known as thermosonication, manosonication, and manothermosonication. The available literature is reviewed, and critically discussed, and potential research gaps are identified. Additionally, discussions on the US's inactivation mechanisms and lethal effects are included. Finally, mathematical modeling approaches of microbial inactivation kinetics due to US-based processing technologies are also outlined. Overall, this review focuses only on the uses of the US and its combinations with other processes relevant to microbial food decontamination.

Growth behavior of low populations of Listeria monocytogenes on fresh-cut mango, melon and papaya under different storage temperatures.

The growth behavior of Listeria monocytogenes low population (1-4 cells/sample) on fresh-cut mango, melon, papaya and fruit mix stored at 4, 8, 12 and 16 °C was evaluated over 10 days. Mango showed the lowest counts for L. monocytogenes during 10 days regardless of storage temperature (<1.7 log cfu.g-1). Melon supported high bacterial growth over 10 days, reaching 5 log cfu.g-1 at 16 °C. Both the fruit and storage temperature influenced the Listeria low population growth potential (δ). Cumulative frequency distribution of L. monocytogenes showed that after 10 days, 100% of fresh-cut fruits and fruit mix stored at 4 °C remained ≤2 log cfu.g-1, while at 12 and 16 °C 100% of melon, papaya and fruit mix samples exceeded this limit. At 8 °C, 100% of mango and fruit mix samples remained below this limit after 10 days, whereas 100% of melon and papaya reached it after 7 days. Results indicate 4 °C as the ideal to store safely fresh-cut mango, melon, papaya and fruit mix for 10 days. Besides, 8 °C can also be an option, but not for melon and papaya. Findings highlight the ability of L. monocytogenes to survive and grow in fresh-cut fruits even at a very low initial population levels.

HPLC Analysis of Phenolic Compounds and Flavonoids with Overlapping Peaks.

The identification and quantification of phenolic compounds and flavonoids in various natural food products is typically conducted using HPLC analysis. Their analysis is particularly complex since most natural food products contain a large number of different phenolic compounds, many of which have similar chemical characteristics such as polarity, which makes complete separation of all eluents extremely difficult. In this work we present and validate a method for the quantitative determination of the concentration of two compounds with similar retention times, i.e. they show overlapping peaks in a mixed solution. Two pairs of phenolic compounds were investigated: caffeic and vanillic acids and ferulic and p-coumaric acids. This technique takes advantage of the different absorbances of the two phenolic compounds in the eluent at various wavelengths and can be used for the quantitative determination of the concentration of these compounds even if they are not separated in the HPLC column. The presented method could be used to interpret the results of HPLC analysis of food products which possess a vast spectrum of phenolic compounds and flavonoids.

Modelling the growth of pear postharvest fungal isolates at different temperatures.

The effect of temperature on the mycelium growth kinetics of four postharvest fungal isolates (i.e., Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer) was assessed. A cardinal model with inflection (CMI) was used to describe the effect of the temperature on the growth rate (µ) and the lag time (λ) of each isolate. Cardinal temperature values such as Tmin, Tmax and Topt were estimated and isolates were sorted according to their growth rate and lag time duration. Additionally, model validation was performed on a medium prepared from mashed pear pulp and on artificially wound-inoculated pear fruits. P. expansum was shown to be the most psychotrophic fungus with the lowest estimated Tmin = -8.78. Model validation on pear pulp agar showed growth rate over-prediction in the case of R. stolonifer and B. cinerea but a good correlation in the case of P. expansum and A. alternata. In vivo experiments on pear fruits showed discrepancies from the synthetic and the simulated counterparts for all the fungi with the only exception of P. expansum.

Investigation of Acrylic Resin Disinfection Using Chemicals and Ultrasound.

PURPOSE: Dental prosthetic and orthodontic appliances are transported from the clinic to the laboratory for additions and repairs. These appliances, containing microbes from the oral flora, are a high risk for cross-contamination. The aim of this study was to evaluate the effect of chemical and ultrasound disinfection against two in vitro biofilms and an in vivo formed biofilm grown on unprepared and polished polymethyl methacrylate (PMMA) surfaces. MATERIALS AND METHODS: Rough and polished self-curing PMMA surfaces were infected with strains of both Candida albicans and Streptococcus oralis. After incubation, the samples were treated with different disinfection methods, including ultrasound treatment for both 15 and 30 seconds, and immersion in glutaraldehyde and alcohol-based chemical disinfectants (MD520 and Minuten, respectively). The disinfecting efficacy was assessed by colony forming units (CFU) analysis and by scanning electron microscopy (SEM). Furthermore the adequacy of bacterial elimination of application of 30-second ultrasound and MD520 was assessed on PMMA retrieved from ten volunteers by CFU analyses. ANOVA with p = 0.05 followed by the Tukey post hoc test and the Student t-test was used to analyze the data. RESULTS: The ultrasound treatment for 30 seconds, MD520, and Minuten were the most effective disinfectant methods as they reduced the microbial counts compared to the control (p < 0.05) as shown in the in vitro analyses. S. oralis adhered more to rough acrylic resin surfaces (p < 0.05). Ultrasound treatment was the most effective way to reduce microbial counts on PMMA exposed to oral flora (p = 0.043). CONCLUSION: Ultrasound treatment for 30 seconds was effective against C. albicans, S. oralis, and the oral flora as shown by testing microbial growth on agar plates and SEM.

A novel model to assess the efficacy of steam surface pasteurization of cooked surimi gels inoculated with realistic levels of Listeria innocua.

Steam surface pasteurization is a promising decontamination technology for reducing pathogenic bacteria in different stages of food production. The effect of the artificial inoculation type and initial microbial load, however, has not been thoroughly assessed in the context of inactivation studies. In order to optimize the efficacy of the technology, the aim of this study was to design and validate a model system for steam surface pasteurization, assessing different inoculation methods and realistic microbial levels. More specifically, the response of Listeria innocua, a surrogate organism of Listeria monocytogenes, on a model fish product, and the effect of different inoculation levels following treatments with a steam surface pasteurization system was investigated. The variation in the resulting inoculation level on the samples was too large (77%) for the contact inoculation procedure to be further considered. In contrast, the variation of a drop inoculation procedure was 17%. Inoculation with high levels showed a rapid 1-2 log decrease after 3-5 s, and then no further inactivation beyond 20 s. A low level inoculation study was performed by analysing the treated samples using a novel contact plating approach, which can be performed without sample homogenization and dilution. Using logistic regression, results from this method were used to model the binary responses of Listeria on surfaces with realistic inoculation levels. According to this model, a treatment time of 23 s will result in a 1 log reduction (for P = 0.1).

Exploring hydrodynamic cavitation for citrus waste valorisation in Malta: from beverage enhancement to potato sprouting suppression and water remediation.

Introduction: The endorsement of circular economy, zero-waste, and sustainable development by the EU and UN has promoted non-thermal technologies in agro-food and health industries. While northern European countries rapidly integrate these technologies, their implementation in Mediterranean food-supply chains remains uncertain. Aims: We evaluated the usefulness of hydrodynamic cavitation (HC) for valorizing orange peel waste in the fresh orange juice supply chain of the Maltese Islands. Method: We assessed: a) the effectiveness of HC in extracting bioactive compounds from orange peels (Citrus sinensis) in water (35°C) and 70% (v/v) ethanol (-10°C) over time, compared to conventional maceration, and b) the potato sprouting-suppression and biosorbent potential of the processed peel for copper, nitrate, and nitrite binding. Results: Prolonged HC-assisted extractions in water (high cavitation numbers), damaged and/or oxidized bioactive compounds, with flavonoids and ascorbic acid being more sensitive, whereas cold ethanolic extractions preserved the compounds involved in radical scavenging. HC-processing adequately modified the peel, enabling its use as a potato suppressant and biosorbent for copper, nitrate, and nitrite. Conclusion: Coupling HC-assisted bioactive compound extractions with using leftover peel for potato-sprouting prevention and as biosorbent for water pollutant removal offers a straightforward approach to promoting circular economic practices and sustainable agriculture in Malta.

Insight to the diversity of Photobacterium spp. isolated from European plaice (Pleuronectes platessa) based on phylogenetic analysis, phenotypic characterisation and spoilage potential.

This study aimed to explore the diversity of fifty-four Photobacterium strains isolated from muscle tissue of European plaice (Pleuronectes platessa) caught at different fishing seasons and stored 14-days under various conditions. Single phylogenetic markers (16S rRNA, gapA, gyrB and recA) and multilocus sequence analysis (MLSA) were employed to classify isolates at species level. Furthermore, intra- and interspecies variability in the phenotypic traits, maximum specific growth rate (µmax) and spoilage potential of the Photobacterium isolates were investigated. The isolates were classified into the P. iliopiscarium (53.7 %), P. phosphoreum (40.7 %) and P. piscicola (5.6 %) clades using MLSA. Two housekeeping genes, gyrB and recA, exhibited a consistent phylogenetic relationship with MLSA, suggesting that they might be used as individual phylogenetic markers for the Photobacterium genus. Intra- and interspecies variability in the expression of phenotypic characteristics and the production of trimethylamine (TMA), inosine (HxR), and hypoxanthine (Hx) were observed. A growth optimum temperature for P. iliopiscarium was approximately 20 °C, while those for P. phosphoreum and P. piscicola were closer to 15 °C. All isolates exhibited the highest growth density at 1.5 % NaCl, followed by 0.5 %, 3 %, and 6 % NaCl. However, P. phosphoreum demonstrated a higher NaCl tolerance than the other two species. Although, the high CO2 atmosphere significantly inhibited the growth of all strains at 4 °C, P. phosphoreum and P. piscicola showed higher growth density at 15 °C than P. iliopiscarium. Notably, all strains demonstrated H2S production. The µmax varied considerably within each species, highlighting the significance of strain-level variability. This study demonstrates that P. iliopiscarium and P. piscicola, alongside P. phosphoreum, are efficient TMA-, HxR-, Hx-, and H2S-producers, suggesting their potential contribution to synergistic off-odour generation and spoilage. Moreover, the Photobacterium isolates seem to exhibit diverse adaptations to their environments, resulting in fluctuated growth and spoilage potential. Understanding intra- and interspecies variability will facilitate modelling seafood spoilage in microbial risk assessments and developing targeted hurdles to prolong products' shelf-life.

Quantitative tools in microbial and chemical risk assessment.

The EU-FORA programme 'Quantitative tools in microbial and chemical risk assessment' was dedicated to training on predictive microbiology fundamentals, implementation of different modelling strategies, design of experiments and software tools such as MATLAB, GInaFiT and DMFit. The fellow performed MATLAB training on maximum specific growth rate (µmax) determination according to the Ratkowsky model. GInaFiT training on different models for bacterial inactivation and DMFit training on growth parameters of Vibrio parahaemolyticus were also carried out. Optical density measurements of V. parahaemolyticus bacterial cultures were performed. The obtained kinetics of optical density measurements were used to estimate µmax. Hereafter, Minimum inhibitory concentrations and non-inhibitory concentrations of aminoglycoside antibiotics were estimated based on the quantification of the fractional areas of the optical density vs time. It can be concluded that the results of the quantitative characterisation of V. parahaemolyticus are reliable and can be used for exposure assessments. Also, the turbidimetric assay can be applied for successful estimation of minimum inhibitory concentrations and non-inhibitory concentrations.

Quantitative tools in microbial and chemical risk assessment.

The popularity of biological origin food protection substances is driven by demands from consumers for natural and clean label product, increasing various food-related safety and health concerns and sustainability issues. Lactic acid bacteria (LAB) are most promising because they are a large group of beneficial microorganisms commonly used in food protection due to their ability to inhibit the growth of pathogenic bacteria and enhance food safety. Extensive scientific research has been conducted to understand the mechanisms by which LAB exert their protective effects in various food systems. Even though LAB activity against various food pathogens and spoilers is distinguished, use of cell-free supernatant (CFS) is still under investigation. This report is dedicated to present how qualitative measures can elaborate in new bacteria-origin food additive investigation. As part of the EU-FORA programme, the fellow was involved in the risk assessment tasks and projects which include gaining basic knowledge in predicative microbiology fundamentals, including different types of modelling strategies; delivering essential understanding about experimental design, knowledge in three specific software tools (MATLAB, GInaFiT and DMFit) and gained overall understanding what are the main differences while modelling growth or inactivation models. Secondary activities were included as a way to expand competences beyond qualitative measures to overall all activities done regarding risk assessment and build a strong network of food safety experts and professionals to continue engaging in risk assessment beyond fellowship programme.

Escherichia coli K-12 Transcriptomics for Assessing the Mechanism of Action of High-Power Ultrasound.

An investigation into the mechanisms of action on bacteria involving exposure to stress factors was conducted in this study. The effects of ultrasound on Escherichia coli K-12 MG1655 and its isogenic mutant, ∆gadW, under high power ultrasound treatments (26 kHz) were screened and identified by analysing their transcriptome differences between primary and secondary sequential treatments using RNA-Seq. This also helped to assess any developed protection for cells between different generations. According to our results, 1825 genes of all tested conditions were expressed, playing different roles in the cell. The expression of these genes is associated with DNA damage, cell membrane integrity, and also metabolic effects. The studied strains also showed different differential expressed genes (DEGs), with some genes being directly responsible for defence mechanisms, while others play an indirect effect due to cell damage. A gradual decrease in the expression of the genes, as we moved from just one cycle of ultrasound treatment to sequential treatment, was evident from a heat map analysis of the results. Overall, E. coli K-12 builds a self-protection mechanism by increasing the expression of genes involved in the respiration for increased growth, and production of flagellum and pili. It can be concluded that high power ultrasound is a technology that triggers several different defence mechanisms which directly link to E. coli.

Fungal and Toxin Contaminants in Cereal Grains and Flours: Systematic Review and Meta-Analysis.

Cereal grains serve as the cornerstone of global nutrition, providing a significant portion of humanity's caloric requirements. However, the presence of fungal genera, such Fusarium, Penicillium, Aspergillus, and Alternaria, known for their mycotoxin-producing abilities, presents a significant threat to human health due to the adverse effects of these toxins. The primary objective of this study was to identify the predominant fungal contaminants in cereal grains utilized in breadmaking, as well as in flour and bread. Moreover, a systematic review, including meta-analysis, was conducted on the occurrence and levels of mycotoxins in wheat flour from the years 2013 to 2023. The genera most frequently reported were Fusarium, followed by Penicillium, Aspergillus, and Alternaria. Among the published reports, the majority focused on the analysis of Deoxynivalenol (DON), which garnered twice as many reports compared to those focusing on Aflatoxins, Zearalenone, and Ochratoxin A. The concentration of these toxins, in most cases determined by HPLC-MS/MS or HPLC coupled with a fluorescence detector (FLD), was occasionally observed to exceed the maximum limits established by national and/or international authorities. The prevalence of mycotoxins in flour samples from the European Union (EU) and China, as well as in foods intended for infants, exhibited a significant reduction compared to other commercial flours assessed by a meta-analysis investigation.

FeMn and FeMnAg biodegradable alloys: An in vitro and in vivo investigation.

Iron-based biodegradable metal bone graft substitutes are in their infancy but promise to fill bone defects that arise after incidents such as trauma and revision arthroplasty surgery. Before clinical use however, a better understanding of their in vivo biodegradability, potential cytotoxicity and biocompatibility is required. In addition, these implants must ideally be able to resist infection, a complication of any implant surgery. In this study there was significant in vitro cytotoxicity caused by pure Fe, FeMn, FeMn1Ag and FeMn5Ag on both human foetal osteoblast (hFOB) and mouse pre-osteoblast (MC3T3-E1) cell lines. In vivo experiments on the other hand showed no signs of ill-effect on GAERS rats with the implanted FeMn, FeMn1Ag and FeMn5Ag pins being removed largely uncorroded. All Fe-alloys showed anti-bacterial performance but most markedly so in the Ag-containing alloys, there is significant bacterial resistance in vitro.

Nanoparticle Food Applications and Their Toxicity: Current Trends and Needs in Risk Assessment Strategies.

ABSTRACT: Nanotechnology has developed into one of the most groundbreaking scientific fields in the last few decades because it exploits the enhanced reactivity of materials at the atomic scale. The current classification of nanoparticles (NPs) used in foods is outlined in relation to the production and physicochemical characteristics. This review aims to concisely present the most popular and widely used inorganic and organic NPs in food industries. Considering that the toxicity of NPs is often associated with chemical reactivity, a series of in vitro toxicity studies are also summarized, integrating information on the type of NP studies and reported specifications, type of cells used, exposure conditions, and assessed end points. The important role of the digestive system in the absorption and distribution of nanoformulated foods within the body and how this affects the resultant cytotoxicity. Examples of how NPs and their accumulation within different organs are presented in relation to the consumption of specific foods. Finally, the role of developing human health risk assessments to characterize both the potential impact of the hazard and the likelihood or level of human exposure is outlined. Uncertainties exist around risk and exposure assessments of NPs due to limited information on several aspects, including toxicity, behavior, and bioaccumulation. Overall, this review presents current trends and needs for future assessments in toxicity evaluation to ensure the safe application of NPs in the food industry.

Principal component analysis of hyperspectral data for early detection of mould in cheeselets.

The application of non-destructive process analytical technologies in the area of food science got a lot of attention the past years. In this work we used hyperspectral imaging to detect mould on milk agar and cheese. Principal component analysis is applied to hyperspectral data to localise and visualise mycelia on the samples' surface. It is also shown that the PCA loadings obtained from a set of training samples can be applied to hyperspectral data from new test samples to detect the presence of mould on these. For both the agar and cheeselets, the first three principal components contained more than 99 % of the total variance. The spatial projection of the second principal component highlights the presence of mould on cheeselets. The proposed analysis methods can be adopted in industry to detect mould on cheeselets at an early stage and with further testing this application may also be extended to other food products.

Aqueous and gaseous plasma applications for the treatment of mung bean seeds.

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Germination assessments were performed in a test tube set-up filled with glass beads and the produced irrigation water. Overall, it was found that the combined seed treatment with direct air CAP (350 W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for first time that aside from the stimulatory effect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can significantly enhance seedling growth. The positive outcome and further applications of different forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research.

Bacterial and fungal contaminants in caprine and ovine cheese: A meta-analysis assessment.

The dairy industry is of great importance to the European economy contributing towards € 8.7 billion of the total trade surplus. Caprine and ovine milk amount to 3.1% of the 152 million tonnes of milk produced in Europe, 95% of which is transformed into dairy products such as cheese. This cheese is mostly produced in small holdings from untreated milk, making it a high-risk dairy product for human consumption. A total of 49 foodborne disease outbreaks caused by dairy products were registered in 2017 in Europe. Therefore, these products remain a serious health risk. This meta-analysis examined 30 studies assessing bacterial or fungal contamination of caprine or ovine milk cheeses. The significantly contaminating microbes were found to be Acremonium spp. (19%), Aspergillus spp. (23%), Bacillus spp. (2%), Brucella spp. (34%), Enterobactericae spp. (36%), Enterococcus spp. (28%), Escherichia spp. (15%), Fusarium spp. (21%), Geotrichum spp. (22%), Listeria spp. (11%), Mucor spp. (15%), Penicillium spp. (25%), Phoma spp. (20%), Rhizopus spp. (15%), Salmonella spp. (3%), Scopulariopsis spp. (19%) and Staphylococcus spp. (25%) in caprine and ovine cheese, indicating a variety of food pathogens as well as spoilers. Raw milk is nutritious hence prone to contamination. However, since traditional cheese is often made from untreated milk, it is important to educate cheesemakers of key safety measures and good manufacturing practice allowing for the safe production of these food items.

Characterization of Indigenous Lactic Acid Bacteria in Cow Milk of the Maltese Islands: A Geographical and Seasonal Assessment.

A geographical and seasonal assessment of indigenous lactic acid bacteria (LAB) in Maltese cow milk was conducted in this study. To investigate this, milk was collected from different regions of Malta during winter and summer seasons. Total viable counts (TVC) and LAB population were enumerated. Afterwards, LAB were isolated and identified by molecular methods. According to the results, similar TVC were enumerated on winter and summer samples, while highest LAB population was detected on summer samples. LAB isolates were grouped in seven different clusters which were assigned to Lactobacillus casei, Pediococcus pentosaceus, Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus rhamnosus, Lactococcus lactis, and Lactococcus garvieae. In addition, Enterococcus and Streptococcus species were also isolated. Season seemed to affect the genus / species of LAB since Lactobacillus were mainly isolated from winter samples, while Lactococcus and Enterococcus species were the main genera identified in summer samples. Regarding the geographical distribution, the majority of the Lactobacillus spp. were isolated from the South-eastern region in both seasons. In conclusion, through this study the diversity of indigenous LAB in the Maltese cow milk was monitored for the first time and highlighted that the microbial communities are affected by seasonality and geographical distribution of the farms.

Characterization of Fungal Surface Contaminants of the Small Maltese June Pear, Pyrus communis var. bambinella.

ABSTRACT: Fungal pathogens cause surface contamination and potential premature fruit spoilage of bambinella, a fruit endemic to the Maltese islands, leading to the loss of fruit during the postharvest phase. The objective of this study was to isolate, quantify, and characterize fungal contaminants of the small Maltese June Pear and describe their growth kinetics. In total, 284 fungicide-free fruits were collected over three consecutive summers (2014, 2015, 2016). The isolated fungi were identified by using forward and reverse colonial morphology. Species identification was determined using PCR-based methods. The number of CFU per square centimeter of bambinella outer skin was calculated. Mycelium diameter growth rate studies of the isolates were also carried out at seven different temperatures, ranging from 5 to 35°C. Fungi isolated from bambinella included Cladosporium ramotenellum, Alternaria arborescens, Penicillium lanosum, Penicillium expansum, and Aspergillus sydowii, listed from the most abundant to the least abundant. The Rosso model was fitted to the growth kinetic data and showed that the optimal temperatures for growth of all five fungi were in the range of 20 to 22°C, whereas growth was slower at temperatures below 10°C and above 30°C. As observed in the diameter studies, the order of highest to lowest germination rate was found to be P. expansum, A. sydowii, P. lanosum, C. ramotenellum, and A. arborescens. Germination studies showed that the highest germination rate was observed for P. lanosum, followed by A. arborescens, C. ramotenellum, P. expansum, and A. sydowii, in descending order. The highest germination lag time was observed for A. arborescens, followed by C. ramotenellum, P. expansum, P. lanosum, and A. sydowii, in ascending order.