Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Activation of Bacillus spores at moderately elevated temperatures (30–33 °C)

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30–33 °C, compared to spores stored at 3–8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.     

Lyophilization, vacuum drying, and subsequent storage of Nosema pyrausta spores

Methods of preserving Nosema pyrausta spores were developed by subjecting laboratoryreared, nondiapausing larvae of the European corn borer, Ostrinia nubilalis, infected with N. pyrausta to different techniques of lyophilization and vacuum drying. Such material was still viable when it was stored in an airtight container and held at ?12°C for 12 months, or when it was stored in a vacuum desiceator and held at 22–24°C for 6 months.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

<Emphasis Type="Italic">Polytrichum commune</Emphasis> spores nucleate ice and associated microorganisms increase the temperature of ice nucleation activity onset

Moss spores disperse via wind and have been found previously in precipitation and air samples. Their presence in the atmosphere led to this study’s examining the potential of moss spores to contribute to ice nucleation, a process necessary for ice formation in clouds prior to precipitation. Ice nucleation assays were conducted using Polytrichum commune spores that were either associated with natural assemblages of microbes or extracted aseptically from capsules and subsequently confirmed to be free of culturable microbes. Liquid suspensions of capsule spores and non-sterile spores nucleated ice at temperatures as high as ?12 and ?7 °C, respectively. When capsule and non-sterile spores were heated at 95 °C for 10 min, which killed all culturable microbes on non-sterile spores, both nucleated ice from ?10 to ?13 °C. An additional non-sterile spore sample collected from partially opened capsules in a forested ecosystem (ID, USA) nucleated ice at temperatures as high as ?7 °C, similar to non-sterile P. commune spores. This is the first set of results to indicate that P. commune spores themselves are capable of nucleating ice at temperatures higher than many abiological particles such as mineral dust (≤?15 °C) and that natural assemblages of microbes can increase their ice nucleation efficiency. Future studies aimed at determining the abundance of moss spores in the atmosphere and the identity of ice-nucleating microbes associated with them will provide further insights into the ability of moss spores to impact precipitation patterns.     

Effect of prolonged storage of spores on field applications of Nosema locustae (Microsporida: Nosematidae) against grasshoppers

Spores of Nosema locustae that were freshly prepared, stored in water at ?10°C for 8 months to 3 years, or stored in cadavers for 1 year at ?10°C were applied on bran at the rate of 10⁹ spores/1.5 lb bran per acre. Applications of fresh spores resulted in higher density reductions and higher incidence of infection among survivors than applications of spores stored in water or cadavers in both a complex of grasshoppers predominated by a single species and in a mixed species complex. Density reductions due to treatment with fresh spores were similar in the two populations, but the mixed species complex had a lower incidence of infection than the complex in which one species predominated. Applications of fresh spores reduced grasshopper densities in both complexes to levels below the economic thresholds.     

Susceptibility of the cigarette beetle Lasioderma serricorne (Coleoptera: Anobiidae) to hypoxia

The susceptibility of the cigarette beetle Lasioderma serricorne (F.) to hypoxia was examined at three different oxygen concentrations (0.5?C0.8, 1.0?C1.3, and 2.0?C2.3?%) and four different temperature/humidity (RH) conditions: 30?°C/75?% RH, 25?°C/75?% RH, 20?°C/43?% RH, and 15?°C/43?% RH. The influence of humidity on mortality was also examined at three humidity levels (21, 43, and 75?% RH) at 1.0?C1.3?% oxygen (O₂) and 25?°C. Our results revealed that adult beetles were the most tolerant at 2.0?C2.3?% O₂ and that the larvae were the most tolerant at O₂ levels <1.0?C1.3?%. Mortality increased with increasing temperatures and decreasing O₂ concentrations. At 30?°C, 75?% RH, and 0.5?C0.8?% O₂, the 99?% lethality (LT₉₉) of larvae was 6.9?days; however, it increased to 20?days when the temperature was decreased to 25?°C or when O₂ levels were increased to 1.0?C1.3?%. Humidity also influenced mortality of both larval and adult beetles. LT₉₉ values for larvae at 25?°C and 1.0?C1.3?% O₂ were 24.0, 44.6, and 50.2?days at 21, 43, and 75?% RH, respectively. Results of this study indicate that a controlled atmosphere (CA) with reduced oxygen levels (<0.5?C0.8?% O₂) represents an effective measure for disinfesting stored tobacco as an alternative to conventional phosphine fumigation at temperatures >30?°C.     

Development of Geocoris varius and G. proteus (Hemiptera: Geocoridae) provided with Ephestia kuehniella (Lepidoptera: Pyralidae) eggs

We studied the development of Geocoris varius (Uhler) and Geocoris proteus Distant reared on Ephestia kuehniella Zeller eggs at 20, 24, 26, 30, 33, or 36?°C. The lower developmental thresholds (T ₀) and the thermal constants (K) of eggs and nymphs of G. varius were 13.3?°C, 151.1 degree-days and 13.4?°C, 433.0 degree-days, respectively; those of G. proteus were 16.1?°C, 98.3 degree-days and 16.9?°C, 226.9 degree-days, respectively. The hatch rate of G. varius eggs was significantly lower at 33?°C than at ??30?°C, and no eggs hatched at 36?°C. That of G. proteus was lowest at 20?°C and did not decline significantly at 36?°C. The survival rate throughout the nymphal period increased with temperature up to 30?°C in G. varius, and it was lowest at 20?°C in G. proteus. Thus, the optimal rearing temperatures for immature stages appear to be about 24?C30?°C for G. varius and 26?C33?°C for G. proteus. It might be possible to improve the efficiency of their mass production by controlling the rearing temperature in the above ranges. This would also make the developmental stages of nymphs more uniform and so prevent cannibalism in mass rearing.     

Osmotic and Desiccation Tolerance in Escherichia coli O157:H7 Requires rpoS (σ38)

The contribution of RecA, Dps, and RpoS to survival of Escherichia coli O157:H7 during desiccation and osmotic stress was determined in Luria–Bertani broth with 12?% NaCl (LB-12) at 30 and 37?°C, on filter disks at 23 and 30?°C, and in sterile bovine feces at 30?°C. RecA did not significantly contribute to survival in any condition or temperature. The contribution of Dps to survival was only significant in LB-12 at 37?°C. RpoS was necessary for survival during desiccation and osmotic stress, and survival of the RpoS mutant was significantly less than the parent in all conditions and temperatures. The RpoS mutant survived up to 21?days in bovine feces,?<4?days on filter disks, and?>8 and?<4?days in LB-12 at 30 and 37?°C, respectively. The parent, ΔrecA, dps, and dps/ΔrecA mutant strains survived?>8?days in LB-12,?>28?days on filter disks, and?>28?days in bovine feces. Increased incubation temperatures were associated with decreased survival. E. coli O157:H7 can persist in desiccating and osmotically challenging environments, especially sterile feces, for an extended period time.     

Factors influencing the pathogenicity and development of Mattesia trogodermae infecting Trogoderma glabrum larvae

Factors that regulate development of Mattesia trogodermae in Trogoderma glabrum were defined, and their quantitative effects were determined. The rate of and the extent to which spore formation proceeds is strictly governed by temperature. More spores are produced at 30° than at 25°C and very low numbers of spores are formed when the incubation temperature is 35°C. When insects are incubated at 35°C for 1–10 days and transferred to 30°C for the remainder of the 30-day experiment, spore production capacity gradually declines with increasing time at 35°C. Two hypotheses are proposed for this phenomenon. Larval size also regulates the extent of spore production, larger larvae having greater potential for spore development. This is not influenced by dosage. Spore production in pupae and adults was always retarded.Dosage and environmental conditions which influence the virulence of M. trogodermae were investigated. These studies show that rates of mortality are higher at higher temperatures. Low doses of spores result in longer LT50's than do high doses at 25° and 30°C. No differences in rates of mortality were found between different doses at 35°C.     

Studies on Prolonged Storage of Beauveria bassiana Conidia: Effects of Temperature and Relative Humidity on Conidial Viability and Virulence against Chickpea Borer,Helicoverpa armigera

Unformulated conidia of Beauveria bassiana were stored at five different temperatures (0°, 10°, 20°, 30° and 40°C) at six different relative humidities (RH) (0, 33, 53, 75, 85 and 98%). Conidial viabilities and virulence against third instar larvae of Helicoverpa armigera were determined over a 24‐month period. Conidia survived longest at lower temperatures (0–20°C) and lower RH levels (0–53% RH). At higher temperatures (30–40°C) conidia did not survive. When the temperature was decreased from 30°C to 0°C, at nearly all RH levels the longevity of conidia increased. Conidia remained virulent for third instar larvae of H. armigera under favourable storage conditions for 24 months.     

Patterns of primary spore discharge of Entomophthora spp from the blue green aphid,Acyrthosiphon kondoi

Experiments were conducted to study the effects of time, temperature, and light regime on primary spore formation at 100% RH for the three major pathogens of Acyrothosiphon kondoi. Only small differences were detected between the continuous light and continuous dark regimes. Entomophthora obscura produced between 6 and 10 × 10³ primary spores mostly during the first 48 hr. Total primary spore production was similar at the five temperatures tested from 5° to 25°C. Entomophthora planchoniana produced large numbers of primary spores (about 5 × 10⁴ per aphid) only at temperatures between 10° and 20°C. The majority of primary spores were formed during the first 24 hr. Primary spore production with Entomophthora nr. exitialis ranged from about 10⁵ per aphid at 5° and 10°C to 3 or 4 × 10⁵ at 15° to 25°C, with most spores being formed during the first 48 hr. It is suggested that rainfall is more likely to be important for transmission of E. obscura and E. nr. exitialis than for transmission of E. planchoniana, and that E. obscura is likely to be the most important pathogen during cool or cold weather.     

Germination and mitochondrial damage in spores of Dictyostelium discoideum following supraoptimal heating

Spores of Dictyostelium discoideum may be quantitatively activated with a heat treatment of 45°C for 30 min. Heat activation at either higher temperatures or for longer duration at 45° C resulted in damaged spores. The spores showed an increased postactivation lag time at 23°C and an increased inability to respond to deactivation with 0.2 M sucrose. As the severity of supraoptimal heating increased, a greater percentage of the spores appeared to contain phase dark lesions and to lose viability. Oxygen uptake began to decrease during and after the appearance of the lesions. Using electron microscopy, the phase dark lesions were found to be mitochondria with disrupted cristae.     

Temperature-Dependent Spore Dimorphism in Burenella dimorpha (Microspora: Microsporida)

Burenella dimorpha, a microsporidian parasite of the tropical fire ant, Solenopsis geminata, produces two morphologically distinct types of spores. The binucleate free spores (spores not bound by a pansporoblast membrane) develop normally at temperatures at least as low as 20°C and as high as 32°C. The uninucleate octospores (spores bound in octets by a pansporoblast membrane), however, develop in a restricted range of temperature. Octospores constituted 35.9%± 2.6 of the spores in 25 pupae held at 28°C. Raising the temperature to 30°C reduced octospores to < 1% of the total spore population. Lowering the temperature to 25° or 22°C reduced the octospore population to 8.5%± 6.5 or 0.4 ± 0.5, respectively. Inhibition of octospore development was complete at 20°C. In contrast, the octospores of Vairimorpha necatrix and Vairimorpha plodiae are reported to be abundant at 16°C and 21°C, respectively. The critical event blocked in octospore development may be meiosis, as evidenced by an abundance of binucleate sporonts in the octospore sequence of development, and absence of more advanced sporogonic stages in hosts held at inhibitory temperatures. Free spore size is not affected by temperature although yield may be slightly reduced at elevated temperature.     

Temperature effects on hyphal growth of wood-decay basidiomycetes isolated from Pinus densiflora deadwood

Hyphal growth rates were tested on malt extract agar plates at eight different temperatures (5–40?°C) using 36 isolates of 17 basidiomycete species obtained from Pinus densiflora deadwood in Japan. All isolates of four brown rot species showed optimum growth at 30?°C, whereas the optimum growth temperature of white rot species varied from 20?°C to 30?°C. Analysis using a dataset from four cooler sites showed that brown rot fungi grew more rapidly than white rot fungi at higher temperatures (25?°C, 30?°C, and 35?°C). These results suggest that the hyphal growth of brown rot fungi might be physiologically adapted to higher temperatures than those of white rot fungi among the fungal species inhabiting deadwood of P. densiflora in Japan.     

Contamination levels of Clostridium estertheticum spores that result in gaseous spoilage of vacuum‐packaged chilled beef and lamb meat

Aims: To determine the contamination levels of Cl. estertheticum spores that result in gaseous spoilage of vacuum‐packaged chilled meats, beef and lamb, stored at two different temperatures, ?1·5 and 2°C. Methods and Results: The study consisted of two separate trials using the same processing parameters applied to beef and lamb at two different storage temperatures and six different inoculation concentrations of Cl. estertheticum. A threshold for pack blowing of c. 1 spore per vacuum pack was seen with both beef and lamb stored at ?1·5 and 2°C. These results highlight the detrimental effect that increasing Cl. estertheticum spore inoculum concentration has on the onset of blown pack spoilage for both meat species stored at ?1·5 and 2°C. Conclusions: This study demonstrates that storage temperature is an extremely important parameter influencing the onset of blown pack spoilage and that storing meat at ?1·5°C significantly delays the onset of blown pack spoilage in comparison with storage at 2°C. Significance and Impact of study: The results of this study indicate that 1 Cl. estertheticum spore may present a risk of spoilage, and thus hygienic carcass dressing is critical to keep contamination to a minimum and maximize storage life of the vacuum‐packed chilled product.     

n-3 Fatty acid content in eggs laid by hens fed with marine algae and sardine oil and stored at different times and temperatures

Inclusion of sardine oil (SO) in diets for laying hens significantly increases the n-3 polyunsaturated fatty acids (PUFAs) in the egg, but these are more sensitive to oxidation, so the storage time and temperature can cause a decrease in their concentration. Therefore, the objective of this study was to determine the effect of algae Macrocystis pyrifera, Enteromorpha spp., and Sargassum sinicola on n-3 PUFA contents in eggs from laying hens fed diets supplemented with sardine oil and stored for different times (0, 15, and 30 days) and temperatures (20°C and 4°C), for 8 weeks. One hundred and twenty hens were divided into four treatments: T1 (commercial diet), T2 (2% SO?+?10% M. pyrifera), T3 (2% SO?+?10% Enteromorpha), and T4 (2% SO?+?10% S. sinicola). At the end, 50 eggs per treatment were collected to quantify total lipids and egg n-3 PUFAs at different times (0, 15, and 30 days) and temperatures (20°C and 4°C) of storage. The results were analyzed using a 3?×?3?×?2 factorial design, and Tukey test to compare means (P?<?0.05). The results show that M. pyrifera and S. sinicola had a better effect on eicosapentaenoic acid, while Enteromorpha was better for docosahexaenoic acid. In relation to time and temperature, the content of the fractions analyzed in the three treatments at 15 days/4°C had a lower loss compared with eggs analyzed at day 0/20°C.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores

Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0 °C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable, temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1% in 14 days.     

The effect of high temperatures on spore germination of Ascosphaera aggregata

Vials containing spores of Ascosphaera aggregara were subjected to temperatures of 65°, 75°, 85°, and 95°C for 8-, 16-, and 24-hr periods at each temperature level in order to simulate disinfection heat treatments and determine the effect of temperature on spore viability. Significant (P > 0.0001) differences were noted for spore germination after heat treatment relative to the origin of the spores, the temperatures to which they were exposed, and for the duration of the heat treatment. An analysis of test responses of all isolates demonstrated significant overall differences in germination by geographic location of origin and temperature relative to duration of the treatment, by location and treatment duration relative to temperature, and temperature and duration of treatment relative to the geographic location of origin.