Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

Biological Control of Botrytis cinerea Pers. ex Fr. in Strawberry by Paenibacillus polymyxa (Isolate 18191)

Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea , the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 10⁶, 1 × 10⁷, 1 × 10⁸) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Effects of Host Cell Density on Cell Infection Level in Antheraea eucalypti (Lepidoptera: Saturniidae) Cell Cultures Persistently Infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua , were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 10³, 5.0 × 10³, and 1.0 × 10⁴ cells/cm²). A difference was observed in the spread of N. bombycis Y9101 infection between low-density and higher-density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short-coiled polar tubes from spores germinated intracellularly.     

A cotyledon screen for resistance to scab (Cladosporium cucumerinum) in cucumber (Cucumis sativus) seedlings

An efficient protocol for differentiating cucumber (Cucumis sativus) seedlings resistant and susceptible to scab (Cladosporium cucumerinum) has been developed by examining the interacting factors of spore density and cotyledon growth stage. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 1 day after emergence by placing a 5 μl inoculum droplet (2 × 10⁶ spores/ml) on the newly expanded cotyledons. Seedlings were incubated in a darkened dew chamber for 48 h at 20°C and 100% r.h., and then grown for 6 days at 20°C. Plants could be rated as resistant or susceptible 8 days after inoculation.     

A bacteriological investigation to elucidate the feeding biology of Nais variabilis (Oligochaeta: Naididae)

SUMMARY. The estimated average number of bacteria in the gut of laboratory-cultured Nais variabilis was 100.4 × 10⁴. This compares with values of 6.7 × 10⁴ and 32.8 × 10⁴, respectively, for N. variabilis and N. barbata obtained from a river.
Comparison of the viability of heterotrophic bacteria in the food and gut of N. variabilis indicated a decline in viability of up to 99.6% during the passage of bacteria through the gut. The estimated gut retention time was 39 ± 8 min at 18°C and therefore loss of viability is unlikely to be due to long retention in the gut. Loss of viability is, however, a prerequisite of bacterial digestion by the worm and provides circumstantial evidence that ingested bacteria are used as food.
No conclusive evidence was obtained indicating (a) preferential ingestion of certain kinds of bacteria, (b) that the gut environment was selectively hostile to the particular kinds of bacteria studied or (c) that worms had a specialized gut micro-flora.     

Effect of Verticillium lecanii on biological characteristics and life table of Serangium japonicum (Coleoptera: Coccinellidae), a predator of whiteflies under laboratory conditions

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum , a predator of whiteflies against five different conidial concentrations (1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×10⁸ spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control.     

Multiple oral inoculations with Cryptosporidium parvum as a means of immunization for production of monoclonal antibodies

Abstract Oral immunization of suckling mice with Cryptosporidium parvum results in a humoral response to a limited set of antigens. Six-day-old BALB/c mice were each inoculated orally with 1 × 10⁶ viable oocysts and subsequently administered oral inoculations of 2 × 10⁶ viable oocysts at 30 and 60 days following the primary infection. After 45 days, mice were boosted with 1 × 10⁶ oocysts orally, plus soluble extracts equivalent to 2 × 10⁶ and 1 × 10⁶ oocysts given intravenously and intraperitoneally, respectively. Four days later, splenic lymphocytes were fused to Ag8 myeloma cells. Using this method, we have been able to select for monoclonal antibodies that predominately recognize sporozoite surface and apical complex antigens.     

Experimental Infection of Nosema sp. Ghose in an Unnatural Host Lesioderma sericorne

ABSTRACT. Various doses of a microsporan parasite, Nosema sp., were fed to third and fourth instar larvae of Lesioderma sericorne that infested different types of stored grains. A spore dose of 3 × 10³ spores/individual resulted in a 39% infection rate, reduction in larval and adult weights, and mean spore concentrations of 1.28 ± 0.2 × 10⁸ spores/larva and 1.1 ± 0.2 × 10⁸ spores/adult. At the above dose, mortality was not well marked (about 35% in larvae and 25% in adults). At 3 × 10⁴ spores/individual, the rate of mortality increases to 80% in larvae and 60% in adults. However, more of the pest population (88% of larvae and 73% of adults) died at a dose of 3 × 10⁵ spores/individual. This dose produced mean spore concentrations of 3.91 ± 0.2 × 10⁸ spores/larva and 2.89 ± 0.2 × 10⁸ spores/adult. Insect death was caused by heavy damage to gut epithelia and fat bodies.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

The effect of procaine on the partitioning of plasmid pSC101

Abstract An examination of samples obtained from a commercial fish smoker, using seawater agar with incubation at 4°, 15° and 37°C for up to 28 days, revealed the presence of large bacterial populations in smoked fish. However, initially only low bacterial numbers, i.e., 2 × 10³/g, were present in the muscle of fresh, whole haddock ( Melanogrammus aeglefinus ). With filleting, there was a sudden increase in numbers to 9.2 × 10⁵/g. Yet immediately after smoking, the bacterial populations decreased (5 × 10⁵/g), followed by a gradual increase with storage (e.g., 2 × 10⁶/g after 24 h). Representative colonies were presumptively identified as Acinetobacter, Alcaligenes , coryneforms, Pseudomonas and Vibrio spp.     

Microbial Spoilage of Fresh Refrigerated Beef Liver

The types and numbers of micro-organisms involved in the spoilage of refrigerated beef liver were studied together with pH, hydration and organoleptic changes of the material. Fresh liver harboured a mixed population ( c . 1 × 10⁵ organisms/g) of Gram positive cocci, chromogens and non-chromogens, sporeformers, presumptive coliforms and Gram negative rods. When samples were rejected organoleptically, after 7–10 days at 5°, the contamination attained levels of c . 7–8 × 10⁷ organisms/g. Spoilage was due to souring; the pH fell from 6·3 (fresh liver) to c . 5·9. Lactic acid bacteria were predominant and Gram negative bacteria did not exceed 1·0 × 10⁶ organisms/g. This type of spoilage is explained by the carbohydrate content of c . 5% in liver. The value of pH appears to be a reliable indicator of liver freshness, with a pH of 6·1 indicating incipient spoilage.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.     

A Model System for Testing the Microbiological Stability of Foods Processed in Laminated Flexible Pouches

A suitable time temperature process for packaging small potatoes in flexible pouches is described. Spores of Bacillus stearothermophilus were inoculated onto the surface of peeled potatoes in flexible pouches. These were evacuated, heat sealed and heated in a steam retort modified to allow a water cooking process with an air overpressure of ca. 68·95 kPa (10 lbf/in²). The D and z values determined in the retort were confirmed by parallel heat-resistance tests with spores in glass ampoules held in an oil bath. Heat treatment at 121·1 °C for 20 min in the retort satisfactorily killed test inocula of spores without overcooking the potatoes. Uninoculated peeled potatoes with a natural level of contamination ( ca. 4 x 10³ spores/sealed pouch) were treated in the retort for different times at 121·1 °C and then incubated at 30 °C for 6 months. No pouches heated for 17–25 min showed microbial growth. This procedure may be applied to any type of food if the numbers of naturally occurring heat-resistant contaminants can be related to the number and heat resistance of a suitable test micro-organism.     

OBSERVATIONS ON THE MICROFLORA OF MINCED CHICKEN MEAT IRRADIATED WITH 4 MeV CATHODE RAYS

SUMMARY: Experiments are described in which minced chicken meat, packed anaerobically, was irradiated at room temperature and in the frozen state with a wide range of doses of 4 MeV cathode rays. Sterility was achieved in 14 out of 15 samples which had received 2 × 10⁶ rads or more. Doses of 0·5 and 1·0 × 10⁶ rads allowed survival of a few bacteria/g, usually spore formers. Bacterial counts indicated an approximately logarithmic decrease in numbers at lower doses, while freezing reduced the bactericidal effect.
The storage life at 5° was prolonged only slightly by doses of 5 × 10⁴ and 10 × 10⁴ rads, and highly variable results were obtained with 17·5 × 10⁴ rads. A dose of 25 × 10⁴ rads, however, increased the storage life very considerably. The types of bacteria present initially, and after irradiation with low doses and storage at 5°, were studied. After storage for 12 days or more various types of nonsporing Gram-positive rods were predominant in almost all samples, both control and irradiated. Streptococci were also important where irradiation with 17·5 × 10⁴ and 25 × 10⁴ rads was followed by long storage.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

Attempts to control cabbage root flies Delia radicum L. and Delia floralis (Fall.) (Dipt., Anthomyiidae) with entomopathogenic fungi: laboratory and greenhouse tests

One laboratory and three greenhouse experiments were conducted to study the pathogenicity and efficacy of Finnish isolates of entomopathogenic hyphemycetous fungi against cabbage root flies. In Petri dishes, exposure to 1.5 × 10¹⁰ spores of Metarhizium anisopliae per dish caused 40–50% mortality of undifferentiated second- and third-stage larvae of Delia floralis , and 1 × 10⁷ spores per dish caused 40–50% mortality of Delia radicum larvae. In one greenhouse test, 1 × 10⁸ and 1 × 10⁹ spores of M. anisopliae and Paecilomyces fumosoroseus reduced the root damage of head cabbage by 20–70% compared with untreated controls, although this was not accompanied by significant reductions in the number of pupae. Only M. anisopliae consistently grew out of larvae and pupae of D. floralis during incubation that followed their recovery from the soil at the end of an experiment testing different formulations of M. anisopliae and Beauveria bassiana , but the frequency of the latent infections of the pest by M. anisopliae was not associated with reduced severity of damage to seedlings of head cabbage.     

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat

an immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30°C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1×10³ cfu ml⁻¹ was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.