Use of Coating to Protect Lyophilized Bacillus popilliae from Moisture

Lyophilized cells of Bacillus popilliae were protected from moisture when suspended in pellets of tung oil polymer which were then coated with paraffin wax. The survival of the protected cells at various levels of relative humidity (RH) and under various storage conditions was determined. During 6 months of storage, moisture appeared to have little effect on survival of the cells when the RH level was 22% or less; but, at higher RH levels, survival declined upon storage. Viable cells were recovered when pellets were stored for 3 months at 33% RH, 2 months at 42% RH, 1 month at 50% RH, and 4 days in distilled water. Under field conditions, some cells survived at least 1 week of storage.     

Rates of Drying and Survival of Rhizobium meliloti Strains During Storage at Different Relative Humidities

An investigation was made of the survival of six strains of Rhizobium meliloti filtered on membrane filters and held in atmospheres of controlled relative humidities (RH) of from 0 to 100% at 30°C in the presence of air. The rate of water loss in the desiccator was determined by the humidity-controlling solution used. Drying was accelerated by a mild evacuation of the desiccator during the drying step. Survival rates of R. meliloti strains were much higher after slow drying to 0% RH than immediately after rapid drying. Fast drying (drying period less than 3.4 h) was shown to adversely affect the tolerance to storage at all RH values tested (no survival after 2 to 5 days of storage). When survival during storage was measurable (after slow drying), the optimum RH values for storage were 43% for strains A145 and Wu498, 22 to 43% for strains RCR2011, Wu499, and Ar16, and 83% for strain RCR2004. The most favorable drying periods were 8, 9.2, 14.2, and 50.1 h for the subsequent storage of strain RCR2011 at RH values of 0, 22, 43, and 83%, respectively. The damaging effects of rapid drying on the tolerance of strain RCR2011 to storage at different RH values could be prevented either by rehydration and subsequent slow redrying or incomplete rapid drying followed by slow drying. It is suggested that R. meliloti strains are susceptible to desiccation stresses. However, the quantitative differences among strains appear to be large enough to permit selection with regard to tolerance to desiccation and storage in dried states.     

Survival of Heterodera zeae in Soil in the Field and in the Laboratory

Eggs and (or) second-stage juveniles (J2) inside cysts of Heterodera zeae survived over winter in the field with no detectable mortality at all six depths to 30 cm from which soil samples were collected between corn stubble in the row at 4-8-week intervals. Few or no free J2 were recovered from soil collected in January-April from the top 5 cm, but some were recovered at all samplings from soil collected at greater depths. Emergence of J2 from cysts and numbers of females developing on corn roots in bioassays of cysts increased substantially between January and April. Cyst numbers in a fallow area of the corn field did not decline at any depth to 30 cm during 20 months. Free soil J2, J2 emerged from cysts, and females from the bioassay of cysts were highest at the first soil sampling in July after 10 months of fallow; numbers of nematodes in all three categories declined thereafter, but a few were still detectable after 20 months of fallow. Some cysts were still being recovered after 51 months from naturally infested field soil stored moist in the laboratory at 2 C and 24 C. Females were produced in the bioassays of cysts recovered from soil stored for 38 months at 24 C and for 32 months at 2 C. No free J2 were recovered from soil after 1 month of storage at -18 C, but even after 7 months storage J2 emerged from cysts that were recovered and many females developed in bioassays of those cysts.     

Biodegradation of Aromatic Hydrocarbons in an Extremely Acidic Environment

The potential for biodegradation of aromatic hydrocarbons was evaluated in soil samples recovered along gradients of both contaminant levels and pH values existing downstream of a long-term coal pile storage basin. pH values for areas greatly impacted by runoff from the storage basin were 2.0. Even at such a reduced pH, the indigenous microbial community was metabolically active, showing the ability to oxidize more than 40% of the parent hydrocarbons, naphthalene and toluene, to carbon dioxide and water. Treatment of the soil samples with cycloheximide inhibited mineralization of the aromatic substrates. DNA hybridization analysis indicated that whole-community nucleic acids recovered from these samples did not hybridize with genes, such as nahA, nahG, nahH, todC1C2, and tomA, that encode common enzymes from neutrophilic bacteria. Since these data suggested that the degradation of aromatic compounds may involve a microbial consortium instead of individual acidophilic bacteria, experiments using microorganisms isolated from these samples were initiated. While no defined mixed cultures were able to evolve ¹⁴CO₂ from labeled substrates in these mineralization experiments, an undefined mixed culture including a fungus, a yeast, and several bacteria successfully metabolized approximately 27% of supplied naphthalene after 1 week. This study shows that biodegradation of aromatic hydrocarbons can occur in environments with extremely low pH values.     

Storage of Resting Spores of the Gypsy Moth Fungal Pathogen, Entomophaga maimaiga

The fungal pathogen, Entomophaga maimaiga causes epizootics in populations of the important North American forest defoliator gypsy moth ( Lymantria dispar ). Increasing use of this fungus for biological control is dependent on our ability to produce and manipulate the long-lived overwintering resting spores (azygospores). E. maimaiga resting spores undergo obligate dormancy before germination so we investigated conditions required for survival during dormancy as well as the dynamics of subsequent germination. After formation in the field during summer, resting spores were stored under various moisture levels, temperatures, and with and without soil in the laboratory and field. The following spring, for samples maintained in the field, germination was greatest among resting spores stored in plastic bags containing either moistened paper towels or sterile soil. Resting spores did not require light during storage to subsequently germinate. In the laboratory, only resting spores maintained with either sterile or unsterilized soil at 4°C (but not at 20 or -20°C) germinated the following spring, but at a much lower percentage than most field treatments. To further investigate the effects of relative humidity (RH) during storage, field-collected resting spores were placed at a range of humidities at 4°C. After 9.5 months, resting spore germination was highest at 58% RH and no resting spores stored at 88 or 100% RH germinated. To evaluate the dynamics of infections initiated by resting spores after storage, gypsy moth larvae were exposed to soil containing resting spores that had been collected in the field and stored at 4°C for varying lengths of time. No differences in infection occurred among larvae exposed to fall-collected soil samples stored at 4oC over the winter, versus soil samples collected from the same location the following spring. Springcollected resting spores stored at 4°C did not go into secondary dormancy. At the time that cold storage of soil containing resting spores began in spring, infection among exposed larvae was initiated within a few days after bringing the soil to 15°C. This same pattern was also found for spring-collected resting spore-bearing soil that was assayed after cold storage for 2-7 months. However, after 31-32 months in cold storage, infections started 14-18 days after soil was brought to 15°C, indicating a delay in resting spore activity after prolonged cold storage.     

Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions

Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy. Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined. Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions. The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs. This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion. Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months. No effect on the microbial permeability of membrane was observed during storage. The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus. The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.     

Effect of long-term cold storage of the predatory mite Neoseiulus californicus at high relative humidity on post-storage biological traits

The present study investigated whether long-term cold storage at high relative humidity (RH) affected the quality of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) in terms of its survival and reproduction. For this purpose, we examined biological traits at the end of storage and during the post-storage period. Mated females three?days after adult emergence were stored individually in 1.5-ml vials for 15, 30, 45, 60, or 75?days at 5.0?±?0.3°C and RH of 99?±?0.1% under continuous darkness. At the end of the storage period, 94–100% of females had survived when the storage period was ≤30?days, but percent survival decreased with longer storage. After storage, female survival and oviposition rates were equivalent to un-stored females at 24?±?1°C, RH of 93?±?2%, and a photoperiod of LD 16:8?h. The quality of progeny (hatchability, survival to adulthood, and sex ratio) of stored females was not affected by storage periods as long as 60?days. These results indicate that storage using the tested method can preserve N. californicus for at least 30?days without any degradation.     

Aging of Soybean Seeds in Relation to Metabolism at Different Relative Humidities

The present study was carried out to clarify the relationshipsbetween respiration, moisture content and the aging of dry soybean(Glycine max) seeds under different storage conditions. Ethanolicfermentation increased with increasing relative humidity (RH)during storage. However, at 75% RH which corresponded to a watercontent of 15.2% in seeds, the production of ethanol ceasedafter one month of storage. The ethanol that had accumulatedwithin the container was re-absorbed by the seeds themselvesand disappeared entirely within 3 months, unless the seeds wereexposed to HCN gas. Similar changes were also found for methanol.The activity of mitochondria increased only in seeds storedat 75% RH, it reached a maximum after the third month of storageand then rapidly declined. The amounts of both CoA and acetylCoA in seeds that had been stored for 4 months were far lowerat 75% RH than at 53% RH, and the viability of soybean seedsstarted to decrease after the fourth month at 75% RH. Therefore,a major cause of the rapid deterioration of seeds at 75% RHat room temperature seems to be the rapid development of mitochondrialrespiration, which is accompanied by the consumption of CoAand its acetyl derivatives. By contrast, the slow deteriorationof seeds during long-term storage at 33% to 68% RH and/or atsubzero temperatures seems to be due to the toxic action ofacetaldehyde, an intermediate in ethanolic fermentation, whichcan slowly occur. (Received April 10, 1995; Accepted July 10, 1995)     

Induction of genetic changes in Saccharomyces cerevisiae by partial drying in air of constant relative humidity and by UV

It was investigated whether there was a critical degree of dryness for induction of genetic changes by drying. Saccharomyces cerevisiae cells were dried in air of 0, 33, 53 and 76% relative humidity (RH). The frequencies of mitotic recombination at ade2, of gene conversion at leul, and of gene mutation at can1 were measured in X2447, XS1473 and S288C strains, respectively.After the cells had been dried at 0% RH for 4 h the frequencies of the genetic changes at ade2, leul and can1 were, respectively, 56, 7 and 3.5 times higher than each spontaneous frequency. Induction rates, defined as the frequencies of the induced genetic changes per unit time (1 h) of drying, were greatly decreased with increase in RH. Partial drying in air of 76% RH up to 4 and 8 h induced no genetic change at ade2 and leul, respectively. It was concluded, therefore, that drying at a certain RH between 53 and 76% gave the critical degree of dryness of cells for the induction of the genetic changes. The water contents of cells (g water per g dry material) were 12% at 53% RH and 21% at 76% RH, whereas the water content of native cells was 212%. Removal of a large amount of cellular water had no effect on the induction of the genetic changes.UV sensitivity of partially dried cells of X2447 for the induction of the genetic change at ade2 drastically increased with decrease in RH between 76 and 53%. The drastic change in the UV sensitivity suggested that photochemical reactivity of DNA of chromosome XV, in which the ade2 locus is located, changed between 76 and 53% RH. It seems that the genetic changes were induced only in the low RH region where DNA in vivo had a different photochemical reactivity.     

Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes

Metarhizium anisopliae was grown on six complex mycological media and on three types of rice at three moisture levels to determine the effect of growth substrate on conidial yield, viability, and virulence against mosquitoes immediately after spore maturation and after the storage of conidia at four different temperature-relative humidity (RH) combinations over a 1-year period. Conidial yields varied with the mycological media, but the viability and virulence of conidia against mosquitoes produced on all substrates were similar when spores were stored under the same conditions. The storage conditions were more critical to spore survival and virulence than the substrate upon which conidia were produced. The comparison of rice types for conidial production indicated that conidial yield, viability, and virulence to mosquitoes were more dependent upon the moisture level during growth and on the storage conditions that upon the rice used. The best storage conditions among those tested for the retention of both spore viability and virulence against mosquitoes were 19°C–97% RH and 4°C–0% RH.     

The Preservation of Bacteria and Fungi on anhydrous Silica Gel: an Assessment of Survival over Four years

The preservation method of Perkins (1962) using suspensions in skim-milk was used to preserve 33 bacteria and 22 fungi on anhydrous silica gel. During storage at room temperature, 64% of the bacteria and 77% of the fungi survived 1 year or more. Storage at 4° often increased the survival period c. 2- to 3-fold: 73% of the bacteria and all 12 of the fungi tested at 4° survived > 1 year. At the last testing, 60% of the bacteria and 36% of the fungi were still viable after storage at 4° for periods between 3 and 4 years. The Gram positive bacilli tended to survive the silica gel preservation process better than most Gram negative bacilli. Some factors influencing survival after preservation on silica gel are discussed; the results support the use of a closed storage tube.     

Aerosol Survival of Pasteurella tularensis and the Influence of Relative Humidity

The aerosol survival in air was determined for Pasteurella tularensis live vaccine strain (LVS) as a function of relative humidity (RH). Three different preparations of bacteria were used: (i) liquid suspension of P. tularensis LVS in spent culture medium; (ii) powders of P. tularensis LVS freeze-dried in spent culture fluid; (iii) P. tularensis LVS freeze-dried in spent culture fluid and then reconstituted with distilled water and disseminated as a liquid suspension. Preparation (i) gave greatest survival at high RH and lowest survival at intermediate RH. Preparation (ii), in contrast, gave greatest survival at low RH and minimum survival at 81% RH. Preparation (iii) was the same as preparation (i), i.e., the process of freeze-drying and reconstituting with distilled water before aerosol formation had little or no effect upon aerosol survival as a function of RH. Hence, control of aerosol survival appears to be through the water content of P. tularensis LVS at the moment of aerosol generation rather than the water content of the bacteria in the aerosol phase.     

Stability of Benzocaine Formulated in Commercial Oral Disintegrating Tablet Platforms

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography–mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.     

Actinomycete growth in conditions of low moisture

Actinomycete communities demonstrated a replacement of the generic composition in time as a function of soil moisture. Representatives of the genera Streptomyces, Micromonospora, Actinomadura, Saccharopolyspora, and Microbispora were repeatedly isolated from soil under different moisture conditions (field capacity, maximum molecular capacity, and maximum adsorption capacity). Representatives of some rare genera (Thermomonospora and Kibdelosporangium) were isolated from soil with low moisture levels inhibiting growth of more hydrophilic actinomycetes and bacteria. Spores of some actinomycetes could grow at low relative air humidity (RH) (50 and 67%). The complete growth cycle of all actinomycetes starting from spore germination to sporulation was observed only at RH of 98%.     

Root Colonization by Agrobacterium tumefaciens Is Reduced in cel, attB, attD, and attR Mutants

Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10³ bacteria/cm of root length at the time of inoculation to more than 10⁷ bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.     

Biochemical and physiological studies of certain ticks (Ixodoidea): effects of relative humidity and starvation on the water balance and behavior of adult Argas (Persicargas) arboreus (Argasidae).

The effects of high (96%) and low (0%) relative humidities (RH) and starvation on the total body weight, water content, dry weight content, and humidity responses of unfed adult Argas (Persicargas) arboreus (Argasidae) were determined each 15 days for a period of 105 days. High RH caused an increase of 6.7% in females and 7.2% in males of total body weight after 15 days followed by very slow decrease reaching 4.5% in females and 5.4% in males of initial body weight after 105 days. Low RH caused continuous decrease to 46.2% in females and 53.7% in males of the initial body weight after 105 days. After 105-day starvation at 96% RH, the initial value of the water content increased by 3% in females and 5% in males; at 0% RH this value decreased by 52% in females and 59% in males. The initial value of the dry weight content decreased by 23% in females and 26% in males at 96% RH and by 33% in females and 43% in males at 0% RH. Newly molted adults were hygronegative and at 96% RH remained so. At 0% RH, the hygronegative reaction gradually decreased and changed to hygropositive after 60 days in females and 90 days in males. Survival of 80 control ticks after 105 days was 96% at 96% RH and 54% at 0% RH.     

Enumeration and Localization of N2-Fixing Bacteria Associated with Roots of Spartina alterniflora Loisel

Numbers and possible locations of N₂-fixing bacteria were investigated in roots of Spartina alterniflora Loisel, which support nitrogenase activity in the undisturbed native habitat. N₂-fixing bacteria were recovered in cultures both from S. alterniflora roots and from the surrounding sediment, and they formed a greater proportion of the bacteria recovered from root homogenates than from salt-marsh sediment. N₂-fixing bacteria were recovered in high numbers from the rhizoplane of S. alterniflora after roots were treated with 1 or 5% chloramine-T for 1 h or with 1% NaOCl for 1 or 2 h. Immersing S. alterniflora roots in 5% NaOCl for 1 h was more effective in distinguishing bacteria inside the roots since this treatment nearly eliminated N₂-fixing bacteria recoverable from the rhizoplane, although high numbers of N₂-fixing bacteria were recovered from homogenates of roots treated with 5% NaOCl for 1 h. However, this treatment was less effective with roots of Zea mays L. (Funks G4646) and Sorghum bicolor (L.) Moench (CK-60 A), indicating that techniques to surface sterilize roots should be evaluated for different plants. Bacteria were observed by light and electron microscopy inter- and intracellularly in the cortex and in the aerenchyma of S. alterniflora roots. This study clearly shows that bacteria, including N₂ fixers, colonize the interior of roots of S. alterniflora growing in a Chesapeake Bay, Maryland, salt marsh.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Survival of Salmonella montevideo on Wheat Stored at Constant Relative Humidity

Eleven samples of Ottawa variety hard red winter wheat were inoculated with a standardized suspension of Salmonella montevideo. The contaminated wheat samples were placed in constant relative humidity (RH) chambers held at 25 C. Relative humidities were 7, 11, 22, 33, 43, 53, 62, 75, 84, 92, and 98%. Constant RH at 25 C was maintained with different saturated salt solutions in the sealed chambers. Periodic counts of viable S. montevideo cells per gram of wheat were made over a 28-week sampling period. Viable counts of S. montevideo on wheat held at 7, 11, and 22% RH decreased from an initial 10⁶ cells/g of wheat to a final count of 10⁴ cells/g in each sample. Samples stored at 33, 43, 53, and 62% RH decreased from 10⁶ viable cells/g to 3.6 × 10³, 10³, 10², and 20 viable cells/g, respectively. No viable S. montevideo cells were detected in the samples held at 75, 84, 92, and 98% RH after 22, 16, 26, and 16 weeks, respectively.     

Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage

Background and Aims

Most priming studies have been conducted on commercial seed lots of unspecified uniformity and maturity, and subsequent seed longevity has been reported to both increase and decrease. Here a seed lot of Digitalis purpurea L. with relatively uniform maturity and known history was used to analyse the effects of priming on seed longevity in air-dry storage.

Methods

Seeds collected close to natural dispersal and dried at 15 % relative humidity (RH), 15 °C, were placed into experimental storage (60 % RH, 45 °C) for 14 or 28 d, primed for 48 h at 0, −1, −2, −5, −10 or −15 MPa, re-equilibrated (47 % RH, 20 °C) and then returned to storage. Further seed samples were primed for 2 or 48 h at −1 MPa and either dried at 15 % RH, 15 °C or immediately re-equilibrated for experimental storage. Finally, some seeds were given up to three cycles of experimental storage and priming (48 h at −1 MPa).

Key Results

Priming at −1 MPa had a variable effect on subsequent survival during experimental storage. The shortest lived seeds in the control population showed slightly increased life spans; the longer lived seeds showed reduced life spans. In contrast, seeds first stored for 14 or 28 d before priming had substantially increased life spans. The increase tended to be greatest in the shortest lived fraction of the seed population. Both the period of rehydration and the subsequent drying conditions had significant effects on longevity. Interrupting air-dry storage with additional cycles of priming also increased longevity.

Conclusions

The extent of prior deterioration and the post-priming desiccation environment affect the benefits of priming to the subsequent survival of mature seeds. Rehydration–dehydration treatments may have potential as an adjunct or alternative to the regeneration of seed accessions maintained in gene banks for plant biodiversity conservation or plant breeding.Key words: Digitalis purpurea, priming, re-drying, seed longevity, seed storage, ageing