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.     

Influence of environmental storage relative humidity on biological indicator resistance, viability, and moisture content.

The effect of environmental storage relative humidity (RH) on the moisture content, viability, and moist heat and gaseous ethylene oxide (EO) resistance of biological indicators (BIs) was evaluated. No statistically significant difference was observed between the initial Bacillus stearothermophilus spore population and the spore population of BIs stored at 20 degrees C and 0, 20, 44, of 55% RH or under ambient, 4 degrees C, or -20 degrees C conditions after 12 months. A statistically significant decrease in moist heat resistance from initial starting levels was found for BIs stored at 20 degrees C and either 0 or 20% RH. There was a statistically significant decrease in the B. subtilis BI spore population, compared with initial levels, when the BIs were stored at 20 degrees C and 0% RH concomitant with a significant increase in their EO resistance. BI storage at 20 degrees C and 20 or 44% RH, or under ambient, 4 degrees C, or -20 degrees C conditions, had no significant effect on EO resistance. BIs stored at 20 degrees C and 66% RH demonstrated a significantly lower EO resistance compared with starting levels.     

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)     

Effect of moisture evaporation from diatomaceous earth pellets on storage stability of Steinernema glaseri

Induction of anhydrobiosis and storage stability of entomopathogenic nematodes are influenced by moisture availability. Decreasing moisture content in diatomaceous earth (DE) pellets containing the Steinernema glaseri NJ-43 strain and its effect on survival time and infectivity of the nematode were determined. Pelletisation was performed in a vortex mixer, using DE Celite® 209 as the desiccant material. Pellets were stored at room temperature (23?±?2°C) and high relative humidity (96–100%). Nematode survival and infectivity against last instar greater wax moth, Galleria mellonella, were tested daily. Initial average and average equilibrium moisture content in pellets were 66.7% and 13.6%, respectively, and the infective juveniles mean survival time was 8.8 days. A moisture transfer model based on diffusion and evaporation was evaluated to predict moisture fluctuations within the pellets. We concluded that 84% of variation in S. glaseri infectivity on G. mellonella larvae was explained by the survival of the nematode, whereas 52% of variation in S. glaseri survival was explained by the loss of moisture from the pellets. The moisture transfer model achieved 78% reliability in predicting moisture content and fluctuations. Therefore, the mechanisms of moisture diffusion and evaporation from the surface to the surrounding atmosphere contribute significantly to moisture loss from the pellets.     

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.     

Survival of Lyophilized Bacillus popilliae in Soil

Survival of lyophilized vegetative Bacillus popilliae in dry soil and in soil kept in atmospheres with different relative humidities (RH) was determined. Cells survived for at least 1 year when the RH was 22% or lower, but an RH of 33% or higher reduced viability. At 33% RH, no bacteria were recovered after 5 months of storage; at 42% or above, no bacteria were recovered after 1 month of storage.     

Comparison of application methods to prolong the survival of potential biocontrol bacteria on stored sugar-beet seed

AIMS: To develop bacterial inoculation treatments on sugar-beet seed that will maintain a commercially acceptable degree of viability for a minimum of 4 months storage at ambient temperature. METHODS AND RESULTS: Single rifampicin-resistant (Rif(+)) strains of both Gram-positive and negative bacterial isolates (mostly pseudomonads) were applied in turn to sugar-beet seed in a comparative study by seed soaking, encapsulation in alginate, pelleting using an inoculated peat carrier or seed priming. The treated seed was assessed for bacterial survival over a time course by plating out homogenized samples onto a selective medium. Priming inoculation offered a significant improvement over all the other application strategies tested. After pelleting with fungicides and drying at 40 degrees C, Pseudomonas marginalis/putida P1W1 maintained populations of >6.6 log(10) CFU g(-1) seed during 4 months storage at 15 degrees C. Subsequent experiments verified a stabilized population under these storage conditions with commercial pellets at <7% moisture content. CONCLUSION: An inoculation method was established which allowed the survival on seed of a Gram-negative bacterium at ambient temperature with little loss in viability. SIGNIFICANCE AND IMPACT OF THE STUDY: This has promising implications for the delivery of beneficial bacteria, especially Gram-negative strains, on sugar beet.     

The solution and solid state stability and excipient compatibility of parthenolide in feverfew

The objectives of this research were to evaluate the stability of parthenolide in feverfew solution state and powdered feverfew (solid state), and explore the compatibility between commonly used excipients and parthenolide in feverfew. Feverfew extract solution was diluted with different pH buffers to study the solution stability of parthenolide in feverfew. Powdered feverfew extract was stored under 40 degrees C/0% approximately 75% relative humidities (RH) or 31% RH/5~50 degrees C to study the influence of temperature and relative humidity on the stability of parthenolide in feverfew solid state. Binary mixtures of feverfew powered extract and different excipients were stored at 50 degrees C/ 75% RH for excipient compatibility evaluation. The degradation of parthenolide in feverfew solution appears to fit a typical first-order reaction. Parthenolide is comparatively stable when the environmental pH is in the range of 5 to 7, becoming unstable when pH is less than 3 or more than 7. Parthenolide degradation in feverfew in the solid state does not fit any obvious reaction model. Moisture content and temperature both play important roles affecting the degradation rate. After 6 months of storage, parthenolide in feverfew remains constant at 5 degrees C/31% RH. However, approximately 40% parthenolide in feverfew can be degraded if stored at 50 degrees C/31% RH. When the moisture changed from 0% to 75% RH, the degradation of parthenolide in feverfew increased from 18% to 32% after 6-month storage under 40 degrees C. Parthenolide in feverfew exhibits good compatibility with commonly used excipients under stressed conditions in a 3-week screening study.     

Influence of humidity on production of pelleted fungal inoculum

One of the practical problems in scaling-up the production of fungal inocula for environmental applications is how to provide essential humidity for fungal growth. Pelleted solid substrate was used as a fungal biomass carrier. It was coated with alginate or agar hydrogels that contained mycelial fragments of the white-rot fungi Trametes versicolor or Irpex lacteus. To follow fungal growth and formation of mycelial coat over pelleted substrate, the fluorescein-diacetate hydrolysing activity (FDA) assay and visual inspection were used. Both fungi were able to overgrow the pelleted substrate in 5–6 days, at a relative humidity (RH) of 86.3% or higher. The enrichment of alginate hydrogel with nutrients or coating of pelleted substrate with more hydrophilic agar hydrogel enabled I. lacteus to overgrow the pellets at a lower RH of 83.6%. Fungal inocula produced at lower RH possessed lower final moisture contents and had greater mechanical strength. Conditioning of T. versicolor mycelial fragments, by a 3-h incubation in fresh growth medium, enhanced fungal growth over the pelleted substrate. A mathematical model was used to simulate and to explain moisture distribution in a hydrogel-coated pellet and the formation of mycelial coat, for various conditions of fungal inocula production.     

Biological fitness of Trichoderma atroviride during long-term storage,after production in different culture conditions

Identification of the production and storage factors that affect conidium germination and bioactivity (fitness) will assist the success of biological control agents. Effects of culturing conditions on conidium fitness of Trichoderma atroviride LU132 were examined in different storage conditions over time. Abiotic factors (temperature, nutrients, water activity and pH) during production were studied. Conidia from the culturing regimes which resulted in greatest and least bioactivity against Rhizoctonia solani in dual culture were selected to assess effects of storage conditions on conidium fitness. Fitness of the test conidia was examined after storage at 30°C and at 0% or 50% relative humidity (RH) over 6 months. Fitness declined over time, and the decline was greater for 50% RH than 0% RH, probably through reduced metabolic activity of conidia during long-term storage. Stored conidia were probably affected by dehydration, temperature and other factors such as oxidation, before and during storage, and also by rehydration after storage. The greatest number of conidia and germination percentage resulted from production at 25°C, but greatest bioactivity resulted from those produced at 30°C. No significant effects on bioactivity were detected between the conidium production treatments C?:?N 5?:?1 and C?:?N 160?:?1, indicating that C?:?N ratio in culture medium is not important for conidium survival of T. atroviride.     

Formulating Atoxigenic Aspergillus flavus for Field Release

A procedure was developed to encapsulate mycelia of an atoxigenic strain of Aspergillus flavus in alginate pellets for seeding into agricultural fields in order to reduce aflatoxin contamination via competitive exclusion. Kaolin, a clay filler commonly employed in alginate formulations, was detrimental to pellet performance as measured by spore yield. Corn cob grits, a by-product of the corn industry, was found to be an excellent replacement for kaolin. Of nine nutritive adjuvants tested, wheat gluten improved pellet performance the most, although gluten concentrations above 5% were difficult to process. The best formulation tested consisted of 1% sodium alginate, 5% corn cob grits and 5% wheat gluten. On a 'per gram' basis, this alginate formulation yielded more spores than either A. flavus sclerotia or colonized wheat seed. Pesticides were also tested as adjuvants with potential use for protecting pellets under field conditions. Only one (chloramphenicol) of four tested pesticides (the others were dichloran, rose Bengal and cyfluthrin) reduced pellet sporulation. Formulations with or without pesticide adjuvants retained similar spore yield potential during a 2-year storage at 8 C. However, spore production in stored products lagged behind that of fresh products. At 75% relative humidity (RH), pellet storage stability decreased with increasing temperature from 27 to 42 C. Pellet spore yield at 32 C decreased as RH decreased from 100 to 90%. Sporulation occurred at 90% RH but not at 88% RH. Spore yield varied widely in four field tests, and the cumulative spore yield was inversely correlated (r2= -0.798, P 0.01) with rainfall. The results suggest that alginate pellets may be effective formulations for delivery of atoxigenic A. flavus strains to furrow-irrigated cotton in desert environments, where aflatoxin contamination of cottonseed is most severe.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

Effects of times and storage conditions of Duddingtonia flagrans chlamydospores in sodium alginate pellets on its nematode predatory ability

This study aimed to determine the effects of Duddingtonia flagrans contained in sodium alginate pellets on trichostrongylide larvae under different storage conditions and durations. The in vitro predatory activity of D. flagrans in pellets against trichostrongylide larvae in sheep faeces were assessed at 0, 1, 3, 6, 9, and 12 months after the pellet was stored under four different conditions (i.e. ?20, 4°C, outdoors, and indoors). These results revealed that the numbers of larvae in faeces of sheep treated with pellets containing chlamydospores (treatment groups) were significantly lower than those in the control groups (without chlamydospores) for all trial months under four storage conditions for different durations (p?<?.05). The obtained reduction rates of the infective larvae (L3) in the four treatment groups ranged from 45.62% to 96.73% throughout the entire experiment. The overall mean L3 reduction percentages were 89.22%?±?3.74%, 88.97%?±?1.33%, 68.60%?±?14.31%, and 75.45%?±?13.18% for 4°C refrigeration, ?20°C refrigeration, indoor, and outdoor conditions, respectively. The pellets stored under these storage conditions for a year were provided to sheep for ingestion (in vivo test), and the results showed that the number of recovered larvae in sheep faeces at 24?h after ingestion were significantly lower than that before ingestion. For in vivo test, the L3 reduction percentage in the faeces was 90.99% (?20°C), 74.81% (outdoor), 83.53% (4°C), and 65.60% (indoor). Under the four storage conditions, D. flagrans spores contained in the pellets can maintain their survival ability to a varying degree in a year.     

Core-shell encapsulation formulations to stabilize desiccated Bradyrhizobium against high environmental temperature and humidity

Engineered materials to improve the shelf-life of desiccated microbial strains are needed for cost-effective bioaugmentation strategies. High temperatures and humidity of legume-growing regions challenge long-term cell stabilization at the desiccated state. A thermostable xeroprotectant core and hydrophobic water vapour barrier shell encapsulation technique was developed to protect desiccated cells from the environment. A trehalose core matrix increased the stability of desiccated Bradyrhizobium by three orders of magnitude over 20 days at 32°C and 50% relative humidity (RH) compared to buffer alone; however, the improvement was not deemed sufficient for a shelf-stable bioproduct. We tested common additives (skim milk, albumin, gelatin and dextran) to increase the glass transition temperature of the desiccated product to provide further stabilization. Albumin increased the glass transition temperature of the trehalose-based core by 40°C and stabilized desiccated Bradyrhizobium for 4 months during storage at high temperature (32°C) and moderate humidity (50% RH) with only 1 log loss of viability. Although the albumin-trehalose core provided exceptional protection against high temperature, it was ineffective at higher humidity conditions (75%). We therefore incorporated a paraffin shell, which protected desiccated cells against 75% RH providing proof of concept that core and shell encapsulation is an effective strategy to stabilize desiccated cells.     

Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules improves survival in simulated gastrointestinal conditions

The aim of the present study was to focus on the impact of two different methods and the effects of cryoprotectants on the survival of a probiotic bacterium, Streptococcus phocae PI80, during storage. For the protection of freeze dried cells, the optimal storage conditions were determined with a high survival rate. After the freeze drying process, all cryoprotectants exhibited a protective effect on cell viability at all storage temperatures. High relative cell viability was observed when cells were incubated at ?20°C, which was optimum for the protection of S. phocae PI80. Trehalose was the most promising cryoprotectant at all temperatures during the storage period of bacterial cells. The combination of trehalose + skim milk showed more than 85% survivability compared to other combinations at ?20°C for 60 days. In addition, encapsulation of probiotic cells into alginate-chitosan gel capsules showed better survival of S. phocae cells (5.468 ± 0.15 LogCFU/mL) with high bacteriocin activity at ?20°C for six months. The cell-loaded microcapsules remained stable when treated with simulated gastric and intestinal fluids. After 6 h in vivo treatment, the capsules were found to be broken, releasing the probiotic cells directly into the intestinal system of rats. Therefore, microencapsulation was found to be the most efficient technique, which not only protected the cells for a longer time but also released the cells into the in vivo intestinal system.     

Induction of egg hatching by high humidity in the cicada Cryptotympana facialis

The timing of egg hatching in Cryptotympana facialis was examined in relation to short-term weather conditions. The brief underwater submergence, once a week, of dead twigs bearing cicada egg nests resulted in high hatching rates both at 25 degrees C and under outdoor conditions protected from rainfall. Under outdoor conditions with natural rainfall, most eggs hatched on rainy days. There was a significant positive correlation between the number of hatching eggs and daily relative humidity (RH). When eggs picked from twigs were exposed to various humidity levels, many eggs hatched quickly at higher humidity without direct contact with liquid water. Newly hatched nymphs showed a low tolerance to desiccation; at 81% RH at 25 degrees C, most of them died within 6 h. Under outdoor conditions, most nymphs died within 2 h on sunny days, whereas nymphs survived longer on rainy days. When newly hatched nymphs were released on dry ground, only 24% of them succeeded in burrowing into the soil, and many were killed by ants or desiccation. However, 92% of nymphs released onto wet ground successfully burrowed into it. The direct induction of hatching by high humidity ensures the survival and establishment in the soil of newly hatched nymphs in this species.     

Reduced leaching of the herbicide MCPA after bioaugmentation with a formulated and stored Sphingobium sp.

The use of pesticides on sandy soils and on many non-agricultural areas entails a potentially high risk of water contamination. This study examined leaching of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) after bioaugmentation in sand with differently formulated and stored Sphingobium sp. T51 and at different soil moisture contents. Dry formulations of Sphingobium sp. T51 were achieved by either freeze drying or fluidised bed drying, with high initial cell viability of 67–85 %. Storage stability of T51 cells was related to formulation excipient/carrier and storage conditions. Bacterial viability in the fluidised bed-dried formulations stored at 25 °C under non-vacuum conditions was poor, with losses of at least 97 % within a month. The freeze-dried formulations could be stored substantially longer, with cell survival rates of 50 %, after 6 months of storage at the same temperature under partial vacuum. Formulated and long-term stored Sphingobium cells maintained their MCPA degradation efficacy and reduced MCPA leaching as efficiently as freshly cultivated cells, by at least 73 % when equal amounts of viable cells were used. The importance of soil moisture for practical field bioaugmentation techniques is discussed.     

Studies with dried airborne vaccinia virus in a small environmental chamber

Dried vaccinia virus was found to be very stable in aerosol form in our chamber at a temperature of 75°F and a relative humidity (RH) of 85% when the virus was protected with 1.5% lysine, 1.5% sodium glutamate, 0.5% isoniazid, 0.5% thiourea, and either 2.5% heart infusion broth (HIB) or a combination of 3.75% lactose plus 1.25% raffinose prior to freeze-drying. Utilization of the Environmental chamber technique resulted in (a) selection of two diluents which protected vaccinia virus against the effects of high relative humidity, (b) measurement of the effect of various chemical additives in the diluents, and (c) quantitative measurement of the moisture absorbed by the various dried products.     

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.     

Formation of ochratoxin A and aflatoxins following the use of propionic acid as a grain preservative for storing damp barley

The growth of storage moulds was studied in barley at 22% and approximately 28% moisture content treated with the recommended and reduced commercial doses of propionic acid over a 6 month storage period at 20°C. Experimental sample size was 5 kg barley per lot. Barley was fully protected against the growth ofA. flavus and aflatoxin formation when the recommended dose was applied. However, the treatment was less effective in controlling growth ofP. verrucosum and preventing ochratoxin A formation such that by 4 to 6 months of storage, the fungus had started to develop and toxin had formed even in some of the samples treated with propionic acid. The risk of the development of ochratoxin A during storage increased as the optimum dose was reduced, particularly for barley at 22% moisture content.