Spore Outgrowth Research Articles

Inhibition of Clostridium perfringens Spore Germination and Outgrowth by Lemon Juice and Vinegar Product in Reduced NaCl Roast Beef

Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV1) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1%, 1.5%, or 2%, w/w), blend of sodium pyro- and poly-phosphates (0.3%), and MoStatin LV1 (0%, 2%, or 2.5%) was inoculated with a 3-strain C. perfringens spore cocktail to achieve final spore population of 2.5 to 3.0 log CFU/g. The inoculated products were heat treated and cooled exponentially from 54.4 to 4.4 °C within 6.5, 9, 12, 15, 18, or 21 h. Cooling of roast beef (2.0% NaCl) within 6.5 and 9 h resulted in <1.0 log CFU/g increase in C. perfringens spore germination and outgrowth, whereas reducing the salt concentration to 1.5% and 1.0% resulted in >1.0 log CFU/g increase for cooling times longer than 9 h (1.1 and 2.2 log CFU/g, respectively). Incorporation of MoStatin LV1 into the roast beef formulation minimized the C. perfringens spore germination and outgrowth to <1.0 log CFU/g, regardless of the salt concentration and the cooling time. Cooked, ready-to-eat meat products should be cooled rapidly to reduce the risk of Clostridium perfringens spore germination and outgrowth. Meat processors are reducing the sodium chloride content of the processed meats as a consequence of the dietary recommendations. Sodium chloride reduces the risk of C. perfringens spore germination and outgrowth in meat products. Antimicrobials that contribute minimally to the sodium content of the product should be incorporated into processed meats to assure food safety. Buffered lemon juice and vinegar can be incorporated into meat product formulations to reduce the risk of C. perfringens spore germination and outgrowth during abusive cooling.

Evaluation of Antifouling Paints Based on Chloric Polymer with Nano-Titanium Dioxide Addition in Material Application Engineering

Attachment and biofouling of marine microbes or organisms have influence on the reliability, service life and environmental adaptability of marine ships. Based on the photocatalysis effect of nano-titanium dioxide and the sterilization ability of cuprous oxide, low toxicity and effective marine antifouling paints were prepared with the utilization of excellent performance of chloric polymer. Evaluation of biofouling resistance was carried out by the laboratory method. Fluorescent microscopy observation indicated that the paints had a good capacity of inhibition to growth of diatoms and spores. Results of influence of different coating area and concentration showed the inhibition capacity of nano-titanium dioxide and cuprous oxide in the paints on biological growth respectively and jointly.

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160°C) value of 6.6 ± 0.1min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8% (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Aqueous extracts of a Mars analogue regolith that mimics the Phoenix landing site do not inhibit spore germination or growth of model spacecraft contaminants Bacillus subtilis 168 and Bacillus pumilus SAFR-032

Because Mars is a primary target for life detection and habitability assessment missions, its exploration is also by necessity a Planetary Protection issue. The recent finding of significant levels of perchlorate (ClO4-) in regolith sampled from the Phoenix landing site raises the question of its potential biotoxicity to putative indigenous martian life, microbial forward contaminants from Earth, or future human visitors. To address this issue, an analogue regolith was constructed based on regolith chemistry data from the Phoenix landing site. A Mars Aqueous Regolith Extract (MARE) was prepared from the Phoenix analogue regolith and analyzed by ion chromatography. The MARE contained (mg/L) the cations Na+ (1411±181), Mg2+ (1051±160), Ca2+ (832±125), and K+ (261±29), and the anions SO42-(5911±993), ClO4-(5316±1767), Cl-(171±25) and F− (2.0±0.4). Nitrogen-containing species NO3-(773±113) and NO2-(6.9±2.3) were also present as a result of regolith preparation procedures, but their relevance to Mars is at present unknown. The MARE was tested for potential toxic effects on two model spacecraft contaminants, the spore-forming bacteria Bacillus subtilis strain 168 and Bacillus pumilus strain SAFR-032. In B. subtilis, spore germination and initial vegetative growth (up to ∼5h) was not inhibited in a rich complex medium prepared with the MARE, but growth after 5h was significantly suppressed in medium prepared using the MARE. Both B. subtilis and B. pumilus exhibited significantly higher rates of spore germination and growth in the MARE vs. DW with no additions (likely due to endogenous spore nutrients), but germination and growth was further stimulated by addition of glucose and a combination of buffered inorganic salts (K2HPO4, KH2PO4, (NH4)2SO4, and MgSO4). The data indicate that the aqueous environment in the regolith from the Phoenix landing site containing high levels of perchlorate does not pose a significant barrier to growth of putative forward contaminants such as B. subtilis and B. pumilus under Earth laboratory conditions.

Mapping the structural requirements of inducers and substrates for decarboxylation of weak acid preservatives by the food spoilage mould Aspergillus niger

Moulds are able to cause spoilage in preserved foods through degradation of the preservatives using the Pad-decarboxylation system. This causes, for example, decarboxylation of the preservative sorbic acid to 1,3-pentadiene, a volatile compound with a kerosene-like odour. Neither the natural role of this system nor the range of potential substrates has yet been reported. The Pad-decarboxylation system, encoded by a gene cluster in germinating spores of the mould Aspergillus niger, involves activity by two decarboxylases, PadA1 and OhbA1, and a regulator, SdrA, acting pleiotropically on sorbic acid and cinnamic acid. The structural features of compounds important for the induction of Pad-decarboxylation at both transcriptional and functionality levels were investigated by rtPCR and GCMS. Sorbic and cinnamic acids served as transcriptional inducers but ferulic, coumaric and hexanoic acids did not. 2,3,4,5,6-Pentafluorocinnamic acid was a substrate for the enzyme but had no inducer function; it was used to distinguish induction and competence for decarboxylation in combination with the analogue chemicals. The structural requirements for the substrates of the Pad-decarboxylation system were probed using a variety of sorbic and cinnamic acid analogues. High decarboxylation activity, ~100% conversion of 1mM substrates, required a mono-carboxylic acid with an alkenyl double bond in the trans (E)-configuration at position C2, further unsaturation at C4, and an overall molecular length between 6.5Å and 9Å. Polar groups on the phenyl ring of cinnamic acid abolished activity (no conversion). Furthermore, several compounds were shown to block Pad-decarboxylation. These compounds, primarily aldehyde analogues of active substrates, may serve to reduce food spoilage by moulds such as A. niger. The possible ecological role of Pad-decarboxylation of spore self-inhibitors is unlikely and the most probable role for Pad-decarboxylation is to remove cinnamic acid-type inhibitors from plant material and allow uninhibited germination and growth of mould spores.

Flow cytometry as a tool to assess the effects of gamma radiation on the viability, growth and metabolic activity of fungal spores

Flow cytometry is often used for viability and vitality assessment in bacteria and yeasts. However, its application to the study of fungal spore development is uncommon, probably due to the difficulties in successfully staining these cells.In the current study, we used flow cytometry for the first time to assess the effects of a disinfection treatment on the survival, growth and metabolic activity of fungal spores (Penicillium chrysogenum, Aspergillus nidulans and Aspergillus niger) submitted to gamma radiation (0–15 kGy). The Forward and Side-Scatter parameters of the cytometer were used to assess the differences in size and complexity of particles. Furthermore, two fluorescent dyes were used: Propidium Iodide to assess the membrane integrity and spore viability, in a culture-independent procedure; and Dihydroethidium to measure the changes in metabolic activity of irradiated spores in their first 10 h of growth in a liquid culture medium.Our results support that flow cytometry is a valuable tool in assessing different biological parameters and biocide effects, as it allowed accurate determination of the viability, growth and metabolic activity of gamma-irradiated spores. The fluorescence of Propidium Iodide was 5–7× more intense in unviable spores. The Dihydroethidium fluorescence increase was associated with faster growth. Control and low radiation doses allowed the germination and growth of spores, while higher doses led to growth inhibition and lower fluorescence.

Antifungal Activities of Hedychium Essential Oils and Plant Extracts Against Mycotoxigenic Fungi

Plant-derived antifungal compounds are preferred to chemicals to reduce the risk of toxic effects on humans, livestock, and the environment. Essential oil extracted from rhizomes and plant extracts of ornamental ginger lily (Hedychium spp.) were evaluated for their antifungal activity against two fungi, Aspergillus flavus and Fusarium verticillioides, that produce two major classes of mycotoxins, aflatoxin, and fumonisin, respectively, on several crops. Essential oil or leaf extracts from several Hedychium species and varieties totally inhibited the growth of pre-germinated spores of A. flavus and F. verticillioides. Control of these two fungi by aqueous extracts of rhizome was not effective, although a reduced number of colonies was observed. In view of these results, essential oils or crude leaf extracts from Hedychium spp. need to be considered for the control of mycotoxin-producing fungi under storage conditions.

Survival and germination of Clostridium perfringens spores during heating and cooling of ground pork.

The effect of heating rate on the heat resistance, germination, and outgrowth of Clostridium perfringens spores during cooking of cured ground pork was investigated. Inoculated cured ground pork portions were heated from 20 to 75°C at a rate of 4, 8, or 12°C/h and then held at 75°C for 48 h. No significant differences (P > 0.05) in the heat resistance of C. perfringens spores were observed in cured ground pork heated at 4, 8, or 12°C/h. At heating rates of 8 and 12°C/h, no significant differences in the germination and outgrowth of spores were observed (P > 0.05). However, when pork was heated at 4°C/h, growth of C. perfringens occurred when the temperature of the product was between 44 and 56°C. In another set of experiments, the behavior of C. perfringens spores under temperature abuse conditions was studied in cured and noncured ground pork heated at 4°C/h and then cooled from 54.4 to 7.2°C within 20 h. Temperature abuse during cooling of noncured ground pork resulted in a 2.8-log CFU/g increase in C. perfringens. In cured ground pork, C. perfringens decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3°C and then increased by 0.9 log CFU/g until the product reached 7.2°C. Even when the initial level of C. perfringens spores in cured ground pork was 5 log CFU/g, the final counts after abusive cooling did not exceed 3.4 log CFU/g. These results suggest that there is no risk associated with C. perfringens in cured pork products under the tested conditions.

A molecular method to assess bioburden embedded within silicon-based resins used on modern spacecraft materials

AbstractCurrent assessments of bioburden embedded in spacecraft materials are based on work performed in the Viking era (1970s), and the ability to culture organisms extracted from such materials. To circumvent the limitations of such approaches, DNA-based techniques were evaluated alongside established culturing techniques to determine the recovery and survival of bacterial spores encapsulated in spacecraft-qualified polymer materials. Varying concentrations ofBacillus pumilusSAFR-032 spores were completely embedded in silicone epoxy. An organic dimethylacetamide-based solvent was used to digest the epoxy and spore recovery was evaluated viagyrB-targeted qPCR, direct agar plating, most probably number analysis, and microscopy. Although full-strength solvent was shown to inhibit the germination and/or outgrowth of spores, dilution in excess of 100-fold allowed recovery with no significant decrease in cultivability. Similarly, qPCR (quantitative PCR) detection sensitivities as low as ∼103CFU ml−1were achieved upon removal of inhibitory substances associated with the epoxy and/or solvent. These detection and enumeration methods show promise for use in assessing the embedded bioburden of spacecraft hardware.

Biological Control Potential of &amp;lt;i&amp;gt;Colletotrichum gloeosporioides&amp;lt;/i&amp;gt; for Coffee Senna (&amp;lt;i&amp;gt;Cassia occidentalis&amp;lt;/i&amp;gt;)

A fungal pathogen, Colletotrichum gloeosporioides was isolated from a greenhouse-grown seedling of coffee senna (Cassia occidentalis) and evaluated as a mycoherbicide for that weed. Host range tests revealed that coffee senna, wild senna (C. marilandica), and sicklepod (C. obtusifolia) were also affected by this pathogen, but 35 other crop and weed species, representing 8 botanical families were not affected. The fungus sporulated prolifically on solid and liquid media with maximum spore germination and growth occurring at 20°C - 30°C. Optimal environmental conditions included at least 12 h of free moisture (dew) at 20°C - 30°C. Spray mixtures containing approximately 1.0 × 105 or more conidia·ml–1 gave maximum control when coffee senna seedlings were sprayed until runoff occurred. Coffee senna seedlings that were in the cotyledon to first-leaf growth stage were most susceptible to this pathogen. Weed control efficacy studies under field conditions demonstrated that control of coffee senna was directly proportional to the inoculum concentration applied. Results of these tests suggest that this fungus has potential as a mycoherbicide to control coffee senna, a serious weed in the southeastern U.S.

Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition

Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo or two other genetic loci encoding homologues of polysaccharide synthesis enzymes fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel Gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Inhibitory Effects of Nisin Against Clostridium perfringens Food Poisoning and Nonfood‐Borne Isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.

Potentiality of organotin(IV) compounds in the control of foliar blight disease of wheat (Triticum aestivum) caused by Bipolaris sorokiniana

Three different types of eight organotin(IV) compounds (of which four were newly synthesised) were screened against Bipolaris sorokiniana. The new compounds were characterised by elemental, IR, 1H and 13C NMR spectral analyses. The experiments were carried out in the field and laboratory between the months of November 2006 to March 2007 and November 2007 to March 2008 at Uttar Banga Krishi Viswavidyalaya, Cooch Bihar, W.B., India. All of the organotin(IV) compounds were tested for the toxicity assay against Indian wheat (Triticum aestivum L.), cv. Sonalika. The spore germination and growth of B. sorokiniana and the biochemical changes associated with the induction of resistance by these chemicals were also tested. The influence of the organic groups (attached to tin atom) of the three different types of compounds studied on the fungicidal activity is remarkably distinct. Some of the compounds tested are more active in controlling the fungus than a commonly used commercial product.

Ecophysiological studies of the non-indigenous species Gracilaria vermiculophylla (Rhodophyta) and its abundance patterns in Ria de Aveiro lagoon, Portugal

Gracilaria vermiculophylla is the dominant macrophyte in the Ria de Aveiro lagoon, Portugal (40°38′N, 8°43′W), which is a highly urbanized estuary impacted by oyster cultivation and finfish aquaculture. This study aimed to understand the success of G. vermiculophylla in estuaries by monitoring its abundance and phenology throughout an annual cycle. This species thrives in soft-bottom communities, mostly entangled in the substrate amongst dead bivalve shells and tubes of the polychaete Diopatra neopolitana but it may be also found free-floating; it is present year-round. Carposporophytes were found throughout the year and at all sampling locations. The life history of this taxon was completed in the laboratory. Spore germination and growth success were investigated in a fully factorial designed array of temperatures (5, 10, 15, 20 and 25°C), photoperiods (8, 12 and 16 h day-lengths) and photon flux densities (40 and 100 µmol photons m−2 s−1). Germination occurred under all conditions, being highest at 20°C under the long day treatment. Germination was lowest at 5°C, with no spore survival. The growth rates of the sporelings were affected by the interaction of the three factors tested. These were minimal at 10°C (2.21% ± 1.14 in 8-h days for tetrasporophytes and 2.60% ± 0.57 in 12-h days for gametophytes) and maximal at 20°C and long day treatments for both gametophyte and tetrasporophyte phases (9.96% ± 0.62 and 16.86% ± 0.73, respectively). No differences were found in the growth rates of the mature haploid or diploid phases. Gracilaria vermiculophylla is reproductive throughout the year. It attains high rates of germination and growth success under a wide range of environmental conditions. With such broad tolerances to environmental factors, G. vermiculophylla has a high invasive potential, which helps to explain its current dominance in the Ria de Aveiro lagoon.

CodY regulation is required for full virulence and heme iron acquisition inBacillus anthracis

Capsule and toxin are the major virulence factors of Bacillus anthracis. The B. anthracis pleiotropic regulator CodY activates toxin gene expression by post-translationally regulating the accumulation of the global regulator AtxA. However, the role of CodY on B. anthracis capsulation and virulence of encapsulated strains has been unknown. The role of CodY in B. anthracis virulence was studied in mouse and guinea pig models. Spore outgrowth and dissemination of the vegetative cells was followed in mice by bioluminescent imaging. We also determined the state of capsulation and the iron requirement for growth of the codY mutant. In all models tested, the codY mutant strain was strongly attenuated compared to the wild-type strain and, in mice, also compared to the atxA strain. The disruption of codY did not affect either ex vivo or in vivo capsulation, whereas atxA deletion affected ex vivo capsulation only. The disruption of codY led to a delayed initiation of dissemination but similar kinetics of subsequent spread of the bacilli. The codY mutant cannot grow on heme iron as sole iron source, whereas the parental and complemented strains can. The lack of CodY-mediated transcription weakens virulence by controlling iron acquisition and synthesis of toxin, but without modifying capsulation.

REMOVAL OF BACILLUS ANTHRACIS STERNE SPORE FROM COMMERCIAL UNPASTEURIZED LIQUID EGG WHITE USING CROSS-FLOW MICROFILTRATION*

ABSTRACT The validity of a cross-flow microfiltration (MF) process for separation of an avirulent strain of Bacillus anthracis (BA) spores from commercial unpasteurized liquid egg white (LEW) was examined. Unpasteurized LEW was inoculated with BA to a level of approximately 106 spores/mL and microfiltered using a 1.4-micron ceramic membrane. Permeate flux increased at a rate of 50 L/h m2 per unit pH decrease, which is almost 30 times greater compared with the rate of increase in flux due to a unit increase in temperature. Spore removal efficiency was at least 6.14 log10 spores/mL. In a storage stability study, microfiltered LEW showed no outgrowth of spores for 21 days at 4C. Egg white protein permeation, and hence the functional properties, were unaffected during MF. PRACTICAL APPLICATIONS Bacillus anthracis (BA), a spore-forming bacterium and a potential biothreat agent, could be used as an atypical foodborne pathogen for intentional contamination of the food supply. Thermal pasteurization is effective only for pathogens that are in vegetative forms such as Salmonella, but ineffective for destroying spore-forming pathogens, such as BA. Also, egg proteins' ability to form foam or gel is severely destroyed during pasteurization, since egg albumin is highly heat sensitive. There is a serious need in the agri-industry to develop an effective intervention strategy for removal of B. anthracis from high value foods such as liquid eggs. The current study describes a microfiltration process that is capable of removing almost 99.9999% BA spores from liquid egg white (LEW) without affecting egg white's ability to form foam or gel, and hence may provide an option for safety and security of liquid foods such as LEW.

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M(w)) and fraction of acetylation (F(A)) of the biopolymer. Chitosans of low acetylation (F(A)=0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F(A)=0.48). For the F(A)=0.16 chitosans with medium (56.8kDa) and higher M(w) (98.3kDa), a better (out)growth inhibition was observed compared to low M(w) (10.6kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6kDa) and high M(w) (163.0kDa) chitosans was only minor. In a spore test concentration corresponding to 10(2)-10(3)CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10(8)CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.

Ripe rot of south-eastern Australian wine grapes is caused by two species of Colletotrichum: C. acutatum and C. gloeosporioides with differences in infection and fungicide sensitivity

Background and Aims: Ripe rot of grapes is widespread in subtropical vineyards of south-eastern Australia. We aimed to re-evaluate the causative agent(s), berry susceptibility, fungicide sensitivity and differences in histopathology. Methods and Results: Both Colletotrichum acutatum and C. gloeosporioides were shown to be responsible for ripe rot in three vineyards surveyed in the Hastings Valley, NSW in 2007 and 2009. Observation on detached berries of Vitis vinifera (cv. Cabernet Sauvignon and Chardonnay) revealed that C. acutatum had a faster infection rate than C. gloeosporioides. C. acutatum also formed appressoria and penetrated grape tissue faster than C. gloeosporioides, which produced longer hyphae on the berry surface before penetration. Both C. acutatum and C. gloeosporioides produced acervuli 48 h after inoculation. In contrast, spore germination rates and growth rates on potato dextrose agar were greater for C. gloeosporioides. The two species differed in their sensitivity to the fungicides benomyl, captan and triadimenol. C. acutatum was significantly more sensitive to captan and triadimenol, whereas C. gloeosporioides was more sensitive to benomyl. Conclusions: Both C. acutatum and C. gloeosporioides cause ripe rot of wine grapes in Australia. Subtle differences in the infection process may explain the relative prevalence of the two species. Significance of the Study: This study confirms the involvement of C. acutatum and C. gloeosporioides in ripe rot of grapes in Australia. Knowledge of growth characteristics and fungicide sensitivity of ripe rot pathogens should aid disease management strategies.