A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores     

Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus)

From 1972 to 1974, estimates of the natural larval mortality (> second instar) of elm bark beetles caused by pathogenic organisms were always below 7'5 % of the beetle population. The pathogenic fungus Verticillium lecanii was frequently isolated from field-collected dead larvae, and in the laboratory all larvae were killed in 5 days when exposed to spore concentrations of 4·5 × 10⁶ spores/ml. V. lecanii begins to lose its pathogenicity after prolonged culture on artificial media. The time taken for V. lecanii to kill Scolytus scolytus larvae when exposed to a logarithmic series of spore dilutions from 9·1 × 10⁷/ml to 9·1 × 10³/ml increased with decreasing amounts of inoculum. Even at spore concentrations as low as 9·1 × 10³/ml the mortality of treated larvae was greater than that of untreated individuals. At 100% r.h. all treated larvae were killed over a temperature range of 5–30 °C; those maintained at 25 °C were killed most rapidly and those kept at 5 °C the slowest.     

Nosema whitei,a microsporidan pathogen of some species ofTribolium

The pathogenicity ofNosema whitei was studied using a dose-mortality technique; larvae ofTribolium castaneum were reared for the duration of each experiment in flour mixed with known numbers of spores. The susceptibility of each of the first 5 larval instars was compared. The LD₅₀ (for mortality after 20 days) increased consistently from the first instar (1.8×10⁶ spores/g) to the fifth instar (1.0×10¹⁰ spores/g). The slopes of the probit lines increased consistently as age increased (from b=1.1 to b=3.9). Two factors which reduce the development time ofT. castaneum, high temperature and high humidity, both reduced the pathogenicity ofN. whitei. Thus pathogenicity decreased as the temperature was increased fram 25°C (LD₅₀=4.2×10⁶) through 30°C (LD₅₀=1.3×10⁷) to 35°C (LD₅₀=3.2×10⁶), also pathogenicity decreased consistently as humidity was increased fram 10%, through 30, 50, 70% to 90% R.H. Adults, emerging fromNosema free larvae, became infected only when exposed to a very high dose (2×10¹⁰ spores/g for 14 days from the day of emergence). Infected larvae were treated for 1 hr. at 45°C in an attempt to cure the infection. The infected larvae were not cured, rather the treatment had an adverse alfect on their survival.

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Résumé La pathogénicité deNosema whitei a été étudiée en élevant des larves deT. castaneum dans de la farine mélangée à des quantités connues de spores. La sensibilité des larves diminue uniformément en fonction de l'age; La DL₅₀ varie de 1,8×10⁶/g (1^er stade) à 1,0×10¹⁰ spores/g (5^e stade). Deux facteurs, qui accélèrent le développement deT. castaneum, des températures et des humidités élevées, réduisent tous les deux la pathogénicité deN. whitei. Les adultes ne peuvent être infectés qu'en les exposant à la dose extrêmement élevée de 2×10¹⁰ spores/g. Un traitement par la chaleur (45°C pendant une heure) n'a pas réussi à guérir les larves.

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This work financed by a Science Research Council (U.K.) studentship is based on a thesis submitted for a degree of Ph. D. at the University of Newcastle-upon-Tyne.     

Effects of Leaf Age, Inoculum Dose and Freezing on Development of Chocolate Spot (Botrytis fabae) Lesions on Field Bean (Vicia faba) Leaves

Botrytis fabae spore suspensions containing c. 1, 10, 10², 10³, 10⁴, 10⁵, or 10⁶ spores/ml were used to inoculate 5, 17 or 30-day-old field bean leaves. The percentages of the leaf areas covered by, chocolate spot lesions and the percentages of the leaf areas bearing conidiophores were assessed 1, 6, 12, 14, and 19 days after inoculation. The percentage of the area covered by lesions and the percentage of the area bearing conidiophores (logit-transformed) increased linearly with increasing spore concentration (log₁₀-transformed). The proportions of leaf areas covered by lesions and bearing conidiophores were both greater on 17 and 30-day-old leaves than on 5-day-old leaves. The rate of lesion growth increased with both increasing inoculum dose and increasing leaf age. Generally there was no interaction between the effects of leaf age and the effects of inoculum dose on either lesion growth or sporulation. Two days after inoculation with suspensions of either 10⁴ or 10⁶ spores/ml, 7-day-old leaves grown at 15°C were transferred to –16°C or 2.5°C or kept at 15°C for 4 days. Two days later more spores had been produced on leaves which had been frozen (–16°C) than on, leaves kept at 2.5°C.     

The effect ofMetarhizium anisopliae (DAT F-001) concentration and exposure time on the survival of the subterranean pasture pest,Adoryphorus couloni (Col.: Scarabaeidae)

The virulence of the DAT F-001 isolate ofMetarhizium anisopliae forAdoryphorus couloni was tested by exposing final instar larvae (L3) to concentrations of 10¹ to 10⁷ spores/g in a sand-peat mix for up to 112 days at 20°±2°C. All concentrations were pathogenic to L3 larvae and survival was dependent on concentration and correlated with exposure time. The time to kill 50% of the treated larvae (LT₅₀) ranged from 18.9 days (10⁷ spores/g) to 82.7 days (10¹ spores/g). The effect of DAT F-001 on feeding by L3 larvae was determined by comparing the production and weight of frass pellets/larva/day of untreated control larvae with larvae rolled in sporulating cultures ofM. anisopliae DAT F-001 and DAT F-054 (low virulence). Exposure to, and infection byM. anisopliae DAT F-001 had minimal impact on larval feeding. L3 larvae continued to feed at the same rate as both untreated and DAT F-054 treated larvae virtually until they died.

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Résumé La virulence deM. anisopliae DAT F-001 pourA. couloni a été analysée en exposant des larves L3 à des concentrations de 10¹ à 10⁷ spores/g dans un mélange de sable et de tourbe pendant un maximum de 112 jours à une température de 20°±2°C. Toutes les concentrations sont pathogènes pour les larves L3; la survie dépendait de la concentration et est en corrélation avec le temps d'exposition. Les LT₅₀ sont compris entre 18,9 jours (10⁷ spores/g) et 82,7 jours (10¹ spores/g). L'effet de DAT F-001 sur l'alimentation des larves L3 a été évalué en comparant la production et le poids des fèces/larve/jour des larves non-traitées avec des larves roulées dans des cultures sporulées deM. anisopliae DAT F-001 et DAT F-054 (faible virulence). L'exposition àM. anisopliae DAT F-001 suivie de l'infection a peu d'effet sur l'alimentation des larves. Les larves L3 continuent à s'alimenter au même rythme que les larves non-traitées de DAT F-054, quasiment jusqu'à leur mort.

     

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.     

Influence of temperature and duration of leaf wetness on infection of Acyrthosiphon kondoi with Erynia neoaphidis

Bioassays were carried out to examine the influence of temperature and duration of leaf wetness on the infectivity of an isolate of Erynia neoaphidis for its aphid host Acyrthosiphon kondoi. Preliminary experiments demonstrated that primary spores produced in vitro were as infectious as those formed in vivo. No consistent effect of temperature on infectivity of primary spores could be detected. The time taken to kill an aphid increased as temperature decreased, from 3–5 days at 20 °C to 12–15 days at 8 °C, suggesting a threshold for disease development of 4 °C. Increasing duration of the period of leaf wetness up to 24 h after inoculation increased the final level of infection. At 20 °C, a minimum moisture period of 3 h was required for infection with maximum infection occurring after about 7 h. These times increased slightly at 15 °C but extending to 7 and 16 h respectively at 10 °C. The epizootiological implications of these results are discussed with reference to previously published data on in vivo production of primary spores of E. neoaphidis.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10¹ CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10¹ and 10⁶ CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10⁶ CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions.     

Laboratory experiments on sugar-beet downy mildew (Peronospora farinosa)

The optimum conditions for Peronospora farinosa betae to produce spores were temperature 8–10 °C and relative humidity 90 % or more, but many spores were produced between 5 and 20 °C and between 80 and 90 % R.H. Most spores were formed in darkness after leaves were exposed to light for 6–8 h. Spores survived exposure to 60 % R.H. for up to 5 days, but were soon killed by temperatures above 20 °C. The germination capacity of spores collected from the field was often very small, but this could not be related to the weather. Most seedlings were infected when inoculated at the growing point and incubated in a saturated atmosphere between 3 and 15 °C for at least 8 h.     

OBSERVATIONS ON THE SURVIVAL AND GERMINATION OF RESTING SPORES OF THREE CHAETOCEROS (BACILLARIOPHYCEAE) SPECIES1,2

Resting spores (hypnospores) of Chaetoceros diadema (Ehrenberg) Gran, Chaetoceros vanheurckii Gran, and Chaetoceros didymus Ehrenberg were collected from a large plastic enclosure moored in Saanich Inlet, B.C., Canada. The effects of combinations of temperature and irradiance on the germination of these resting spores were investigated. Nutrient uptake, carbon fixation, and changes in the photosynthetic capacity of the germinating spores were also examined. Resting spores germinated optimally at combinations of temperature and irradiance similar to those in the environment during sporulation. They did not germinate at irradiances 1.3 μEin m^?2 s^?1 or temperatures >25.3° C. Nitrate, phosphate and silicate were taken up after the resting spores had germinated and resumed vegetative growth. Chlorophyll a fluorescence in vivo, and the DCMU-induced increase in in vivo fluorescence also increased after the resting spores had germinated. Resting spores began to fix carbon as soon as they were placed in light. Spores remained viable for at least 645 d. The length of time between first exposure to light and germination did not change during this period; however, the percentage of viable resting spores decreased markedly. None of the Chaetoceros spores germinated after 737 d of storage at 2–4° C in darkness.     

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.     

Moulding of sugar-cane bagasse and its prevention

When fresh sugar-cane bagasse containing about 50% water and 3% sugar was baled and stacked, it quickly heated to over 50°C and remained hotter than 40°C for 50 days. The residual sucrose was utilized by microbial growth and the content of fungal, bacterial and actinomycete spores increased to more than 10⁸/g dry wt. The spores in heated bagasse were mostly of thermophilic actinomycetes and fungi, and included two actinomycetes implicated in bagassosis. Thermoactinomycetes sacchari occurred in 40% of samples examined, some of which yielded up to 5 × 10⁶ colonies/g, while T. vulgaris occurred in 80% of samples, but these rarely yielded more than 10⁵ colonies/g. Other organisms were cellulolytic and caused fibre deterioration. Heating and moulding could be much decreased either by drying to about 25% water content, which halved the spore content after storage, mostly at the expense of the actinomycetes, or by adding 2% by weight of propionic acid, which decreased the spore content to 4 × 10⁶ spores/g or less even after 18 months' storage. Sometimes adding only 0·6% of propionic acid or 2% of propionic acid applied as a 50% aqueous solution had a similar effect. Treatment with propionic acid thus decreased the deterioration of bagasse, permitted its storage between harvests and prevented the hazard of bagassosis to workers.     

Relationship between Position on the Parent Plant and Dormancy Characteristics of Seeds of Three Cultivars of Celery (Apium graveolens)

Germination and seedling emergence studies were made on seeds harvested from four different umbel positions of three cultivars of celery (Apium graveolens L.). Although heavier seeds were produced from primary umbels than from other umbels, these were less viable as measured by the germination percentage at I8°C in the light. However, germination of viable seeds from quaternary umbels was lower than that of seeds from primary umbels at 18°C in the dark when incubated with GA₄, (2 × 10 ^?4M) and seed from secondary and tertiary umbels tended to be intermediate in response. All viable seeds germinated when N⁶-benzyladenine (10^?2M) was used in combination with GA₄. Seeds from quaternary umbels of two of the cultivars had a lower high-temperature limit for germination in the dark than did seeds from other umbels. In glasshouse experiments the emergence of viable ‘quaternary’ seeds of these cultivars was higher than that of ‘primary’ seeds. Under these conditions the time to 50% of the final emergence as determined after 42 days was similar for seeds from all umbel positions within each cultivar. In two varieties seedling weights were greater from seeds of primary as compared to quaternary umbels, and in general, the largest seedlings arose from the heaviest seeds and the smallest from the lightest seeds in all three cultivars.     

Control of Postharvest Diseases of Sweet Cherry with Ethanol and Hot Water

Complete inhibition of the germination of spores of Penicillium expansum occurred after 10 s exposure to 40% ethanol or more at ambient temperature, while spores of Botrytis cinerea were completely inhibited by 30% ethanol or more. Mortality of the spores of P. expansum and B. cinerea in heated 10% ethanol was higher than in water at the same temperatures. Immersion of naturally inoculated fruit in 20, 30, 40, or 50% ethanol reduced the decay present after storage for 10 days at 20°C similarly and by approximately 60–85%. Immersion of fruit that had been inoculated with the spores of P. expansum and B. cinerea reduced decay by both pathogens after storage for 30 days at 0°C and 5 days at 20°C when 30% or higher concentrations of ethanol were used. The incidence of decay after immersion in water alone for 30 s at 24, 50, 55, or 60°C was 57.7, 44.7, 46.2, and 35.7%, respectively, while 10% ethanol at these temperatures the decay incidence to 52.2, 33.9, 32.8, or 14.7%, respectively. Water treatments at 50, 55, or 60°C alone were not effective against P. expansum, while their efficacies were significantly increased by the addition of 10% ethanol. The most effective treatment was immersion in 10% ethanol at 60°C. Ethanol treatments at 20, 30, 40, or 50% and water treatments at 55 or 60°C significantly reduced natural fungal populations on the surfaces of fruit in all of the experiments. Addition of 10% ethanol to water significantly increased the efficacy of water in reducing the fungal populations at elevated temperatures. None of these treatments caused surface injuries to the fruit or adversely affected stem colour.     

Effects of pH,NH<Subscript>4</Subscript>-N concentration,temperature, and storage period on basidiospore germination in an ectomycorrhizal ammonia fungus <Emphasis Type="Italic">Hebeloma vinosophyllum</Emphasis>

Basidiospore germination in an ectomycorrhizal ammonia fungus Hebeloma vinosophyllum was stimulated by 10–500 mM NH₄Cl aqueous solution at pH 4.5–9.0, but not by pure water. The basidiospores germinated at 10°–35°C with an optimum at 25°–30°C. The highest germination percentage (83.0%) was observed in 100 mM NH₄Cl aqueous solution adjusted to pH 8.0 by KOH, when the basidiospores were incubated at a density of 10⁶ spores/ml at 30°C for 14 days. The percent germination value decreased with the increased duration of storage under both dry and wet conditions. Humidity and temperature affected the longevity of H. vinosophyllum basidiospores. The basidiospores maintained their germination ability longer under a dry condition than under a wet condition. The greatest longevity was accomplished by storage at 15°C under a dry condition.     

A cotyledon double inoculation technique for evaluating resistance to anthracnose (Colletotrichum orbiculare) and scab (Cladosporium cucumerinum) in cucumber

A technique for simultaneous inoculation of cucumber cotyledons with Colletotrichum orbiculare race 1 and Cladosporium cucumerinum has been developed. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 24 h after emergence with Colletotrichum orbiculare (200 spores in 2 μ1 of water) and Cladosporium cucumerinum (1000 spores in 5 μ1 of water) followed by 48 h of incubation in the dark at 20°C and 100% r.h., and 48 h in a 20°C lighted growth chamber. Seedlings were then moved to a growth chamber at 21°C at night and at 26°C during the day for 4 days and plants were rated as resistant or susceptible 8 days after inoculation. No interference in the expression of resistance or susceptibility of cultivars to either pathogen was detected in simultaneous inoculations.     

Survival and persistence of nonspore-forming biothreat agents in water

Aims: To determine whether nonspore‐forming biothreat agents can survive and persist in potable water that does not contain a disinfectant. Methods and Results: Autoclaved, de‐chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml^?1). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature. Conclusion: Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore‐forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C. Significance and Impact of the Study: The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.