Shelf-life of a Biocontrol Pseudomonas putida Applied to Sugar Beet Seeds Using Commercial Coatings

Pseudomonas putida 40RNF is a putative biological control agent (BCA) of Pythium damping-off of sugar beet. The survival of 40RNF during commercial seed treatment and its subsequent shelf-life (i.e. long-term viability and biocontrol activity) were assessed. Two methods were used to apply 40RNF to sugar beet seeds: incorporation into film-coats sprayed on to pre-pelleted seeds and incorporation into the pellet material prior to pelleting. Only 7.1% of applied 40RNF survived film-coating, but an initial concentration of 7 × 10⁸ ensured that 83.3% of a pre-determined target rate of 6 × 10⁷ |pellet was achieved. After 52 weeks of storage at 4°C,the numbers of 40RNF had declined by one to two orders of magnitude, with a decrease of approximately 50% in disease control. After 52 weeks at 18-20°C, 40RNF was below detectable limits (< 100|pellet), yet the biocontrol activity of the seed treatments was not reduced. The survival of 40RNF during incorporation into the pellet material was poor (< 0.2% of those applied, i.e. 5 × 10⁵ pellet). However, bacterial viability and biocontrol efficacy were maintained at 100% of the control value for 24 weeks when stored at 18-20°C. The results indicate that commercial seed treatments and the storage of pellets at ambient temperatures has potential for the introduction of bacterial BCAs into the spermosphere.     

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.     

Constant, Fluctuating and Effective Temperature and Seed Longevity: a Tomato (Lycopersicon esculentum Mill.) Exemplar

Tomato seeds with a moisture content of 16.4% were stored hermeticallyat one of five constant temperatures (10, 20, 30, 40, 50 °C)or in one of nine alternating temperature (24 h/24 h) regimes(10/30, 10/40, 10/50, 20/30, 20/40, 20/50, 30/40, 30/50, 40/50°C) for up to 224 d. In each regime, seed survival conformedto cumulative negative normal distributions and all 14 survivalcurves could be constrained to a common origin. Estimates ofthe constants C_Hand C_Qof the viability equation determined atconstant temperatures were 0.0346 (s.e. 0.0058) and 0.000401(s.e. 0.000096), respectively. The effective temperature forseed survival of each alternating temperature regime was alwaysmuch higher than the mean. Tomato seeds were also stored hermeticallyat 15.9% moisture content at 40 °C for 0, 7, 14, 21 or 28d before transfer to 50 °C. This investigation showed thatthe standard deviation of the subsequent survival curves at50 °C was unaffected by the duration of previous storageat 40 °C. The results of both investigations were consistentwith the hypothesis that loss in probit viability is solelya function of the current storage environment, with no effectof change in temperature per se. The application of the viabilityequation to seed survival in fluctuating environments was validatedagainst independent observations for rice in uncontrolled storageconditions. Copyright 2001 Annals of Botany Company Temperature, seed storage, longevity, moisture content, viability equation, tomato, rice     

A study of the longevity of the seed-borne parasites of flax in relation to the storage of the seed

From 1939 to 1947 observations were made on the longevity of Colletotrichumlinicola Pethybr. & Laff., Polyspora Lini Laff., Botrytis cinerea F t ., and Phoma sp. occurring to different extents as seed-borne parasites of naturally contaminated samples of flax seed held under ordinary conditions of storage. The results obtained, together with the germination capacity of the seed estimated on different occasions during storage, are given and show that the longevity of the fungi considered varies within the following limits: Colletotrichum linicola , 26–69 months, Polyspora Lini , 16–55 months, Botrytis cinerea , 16–40 months and Phoma sp. 27–43 months. The data provide grounds for suggesting that the higher the percentage number of seeds contaminated with Polyspora Lini the longer will be the time taken for the death of the parasite to occur. As the time taken for the deactivation of the parasites is normally much longer than that for which flax seed for sowing purposes can be safely stored, prolonged storage of the seed does not commend itself as a likely method for securing the control of the diseases caused by seed-borne fungi. The germination capacity of the seed was normally found to be seriously impaired by the time all the parasites had died.     

Studies on the survival and transmission of Xanthomonas manihotis on cassava seed

Cassava seed which had been stored at 5 ^oC and 60% r.h. for 2–51 months was assayed for the presence of Xanthomonas manihotis by a leaf-infiltration technique, using as inoculum the supernatant from seeds soaked in sterile water at 30 ^oC for 2–4 h. The threshold of sensitivity of the assay method was 10⁵ cells/ml. Twenty out of 50 samples yielded the pathogen. The infested seed had been in storage for 2–18 months. Bacteria reisolated from infiltrated leaves were identical to X. manihotis in cultural characteristics, phage type and pathogenicity. Surface sterilisation or hot air treatment for 24 h at 65^oC or lower did not eliminate the pathogen from infested seed. Soaking of infested seed in hot water at 60 ^oC for 20 min reduced the number of bacteria to less than the minimum detectable level without appreciably reducing germination. Cassava bacterial blight was observed in 8-wk-old seedlings which had been planted during the dry season at a site where infection from outside sources was unlikely. It is postulated that a low percentage of successful seed transmissions of X. manihotis can occur under favourable environmental conditions.     

Systematic overestimation of Salicaceae seed survival using radicle emergence in response to drying and storage: implications for ex situ seed banking

Biodiversity conservation programmes are underpinned by seed banking following drying to low water contents (WC), and supported by both the assessment and prediction of seed viability over time. The means of judging viability is thus crucial to the comprehension of seed vigour. We selected seeds of three species and one hybrid in the Salicaceae likely to have variation in tolerance to drying, processing and storage, including in relation to cryobanking, and compared survival growth as radicle emergence (germination) and normal seedling production. With three seed lots of Salix gracilistyla, air-drying to 8–10 % WC enhanced seed survival after 40 days’ storage at 5 °C as compared with non-treated seeds at 14–20 % WC. Four seed lots of Populus alba × P. glandulosa showed equally high germination (88–100 %) and proportions of normal seedlings (81–99 %) when stored at 5 °C for 7–10 weeks. Among seven seed lots of S. gracilistyla, two groups with different storage behaviour could be statistically distinguished with normal seedling production ranging from 0 to 45 % after storage at 5 °C for 13 weeks. Seed tolerance to WC manipulation and cryopreservation was very variable among species and seed lots. Seed lots of S. hallaisanensis and S. gracilistyla with ~80 % germination survived cryopreservation at 10 % WC, but were sensitive to lower WCs. In contrast, Populus seeds had greater desiccation tolerance combined with cryopreservation capability. With seed lots of all species and hybrids, cryopreservation had little effect on viability unless the high moisture freezing limit had been exceeded (~10–20 % WC, depending on seed lot). However, under all conditions of handling (drying, rehydration, storage at 5 °C or cryopreservation) using germination as the only indicator of viability over-estimated survival compared with normal seedling production.     

Studies of halo-blight seed infection and disease transmission in dwarf beans

The transmission of Pseudomonas phaseolicola from plant to seed was mainly by the penetration of bacteria from external pod lesions to the underlying seeds. There was no evidence for translocation of bacteria from other parts of the plant to the seed, and symptomless pods contained only healthy seeds. Although a small proportion of infected seeds showed obvious symptoms of infection, the majority showed either slight symptoms, which could be detected only by careful observation, or were symptomless. In tests of disease transmission from seed to seedling, seeds with slight symptoms, or those which were symptomless were responsible for 35% and 52% respectively of the total disease transmission compared to 13% for obviously infected seeds. The viability of bacteria in seed stored under relatively uncontrolled conditions (10–27 C) declined by a factor of 250 per annum over the first 3 yr with extrapolation predicting effective extinction after c . 5 yr. The pathogen survived longer under controlled conditions (7–10 C and 45–50% r.h.) but no viable bacteria were detected in seed stocks which were 10-yr-old and with one exception 6-yr-old stocks were also free of the pathogen.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

SEED AND SOIL TREATMENT WITH SYSTEMIC INSECTICIDES TO PREVENT APHID COLONIZATION OF EMERGING BEET SEEDLINGS

Dimefox at 2 lb. or diethyl ethylthiomethyl dithiophosphate (Thimet) at 1 lb. applied at drilling in 100 gal. water along the drills, or seed treated at rates giving 6–8 oz. Thimet or 8–24 oz. diethyl ethylthioethyl dithiophosphate (Disyston) per acre†, made sugar beet seedlings toxic to aphids up to 30 days after sowing four root crops in April-May, and up to 30–40 days after sowing five steckling crops in autumn. Malathion, demeton, demeton methyl, bis (dimethylamino) azido phosphine oxide (N.C.7) and schradan were less effective. The infestation of green aphids was decreased by the treatments during what is often a critical period for virus infection in summer-sown stecklings and occasionally in spring-sown root crops. Germination was 73–100% of the control after soil treatments, 91–98% after Disyston seed treatments and 62–84% after Thimet seed treatments. The treatments slightly decreased Aphis fabae injury to steckling seedlings in 1955 and the number of plants with yellows in a steckling experiment in 1956.     

Celery leaf spot: sources of inoculum

The relative importance of infected celery seed, infected leaf debris in the soil, and infected wild celery, in the incidence of Septoria leaf spot in cultivated celery has been investigated. Infection can be caused when the sole source of inoculum is viable spores on the seed surface; such spores are considered to be the main cause of disease outbreaks. Of all the seed samples examined, 93% were infected by Septoria spp. In untreated seed samples, 40% carried viable spores which survived for up to 15 months on seed stored in the laboratory, and for longer periods on seed stored at -20d? C. However, ageing of seed is not recommended as a commercial control measure. The fungus was not found in seed embryos or endosperms but mycelium was present in pericarps and testas. Unconfirmed evidence suggests that in favourable circumstances new spores might be produced in old seed-borne pycnidia.     

The Survival of Starter Organisms in Concentrated Suspensions

S ummary : Two representative lactic acid bacteria were grown in a rich organic medium, the cells were harvested by centrifugation, and the organisms were suspended in skimmilk at concentrations of 25–55 × 10⁹/ml. The pH was adjusted to different values and the suspensions were stored, with and without the addition of 20% (v/v) glycerol, at 4° and - 20°.
Both organisms survived better at pH 7°0 than at pH 5°0. Glycerol protected the cells at the lower pH value but offered no benefit at neutrality. At 4° about half the cells died within 5 or 6 weeks. At - 20°, however, there was no appreciable loss of viability or acid producing ability up to 9 months at pH 7°0. Suspensions stored for 9 months at - 20° produced excellent cultured buttermilk within the normal incubation period from an inoculum comparable to that used for a fresh culture. Cells harvested early in the maximum stationary phase of the growth cycle were more active than older cells, and they survived better than older cells during storage in the frozen state.     

Persistence and Distribution of Erysipelothrix rhusiopathiae and Bacterial Indicator Organisms on Land Used for Disposal of Piggery Effluent

Erysipelothrix rhusiopathiae was isolated from 15 of 40 effluents collected from commercial piggeries. The organism was isolated from soil and pasture of experimental disposal sites for up to two weeks after application of effluent naturally infected with Ery. rhusiopathiae. The organism was more commonly isolated from soil than pasture. Times for 90% reduction (T₉₀ values) of indicator organisms over a six week period following the effluent applications where Ery. rhusiopathiae was detected for 7 d or more, were 8–19 d for faecal coliforms in top soil and 5–12 d on pasture. T₉₀ for faecal streptococci was 10–14 d in soil and 8–11 d on pasture. Laboratory investigations indicated that the death rate of Ery. rhusiopathiae was up to six times greater than Escherichia coli in soil at field capacity. In dry soil the differences in die-out rate of the two organisms was less marked.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds

Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation.     

Enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum.

The enteropathogenicity of Vibrio parahaemolyticus was investigated by contrasting the effects of whole cells, cell fragments, cell-free preparations, and media constituents injected into rabbit ileal loops. Three of 20 cultures utilized were Kanagawa-negative strains from seawater and sea fish. The remaining 17 cultures included both Kanagawa-positive and -negative strains from Japanese victims of gastroenteritis. Broth culture filtrates concentrated 10-fold by dialysis against 30% Carbowax were unreactive, whereas lyophilized filtrates, regardless of Kanagawa type, as well as all sterile broth preparations containing greater than or equal to 5% NaCl gave positive reactions in the rabbit gut. In contrast, crude lysates derived from broth cultures of Kanagawa-positive strains caused loop dilatation; lysate supernatants were unreactive. Lysates of cells washed from brain heart infusion agar were more reactive than lysates from Trypticase soy agar-grown cells. When agar-grown cell lysates prepared by disruption in saline were dialyzed against distilled water, they were devoid of gut reactivity. Reactivity was restored in dialysands resuspended in saline and in dialysates concentrated 10-fold. The agar-grown cell lysates exhibited Kanagawa-type hemolysis. Our data support the conclusion that the rabbit loop reactivity observed with lyophilized, cell-free culture filtrates may result from excessively elevated NaCl concentrations, and that a toxic factor associated with large-cell particles may be dialyzable, depends on saline for expression, and resembles the Kanagawa hemolysin.     

Fiber development in preanthesis cotton ovules

A tissue culture method was developed to investigate the production of cotton (Gossypium hirsutum L. cv. Texas Marker-1) fibers in vitro. Ovules were excised from 3, 5, 7 and 9 days preanthesis ovaries and placed on an agar-solidified, modified Murashige and Skoog medium containing 2.3 μ M kinetin and 0.45 μ M –2,4–dichlo-rophenoxyacetic acid or 2.3 μ M kinetin and 10.7 μ M naphthaleneacetic acid. Ovules formed fibers and callus tissue. Fibers formed in vitro were up to 10 mm long, 10–22 μ wide and the cell wall was 1–3 μ M thick. Callus tissue cells were subcultured for over 25 weeks and their degree of elongation was monitored. The ability of ovule-derived cells to direct expansion in a longitudinal direction diminished, while lateral expansion increased with time in culture.     

Efficacy of agar media for enumerating two Saccharomyces species in sucrose syrups

Experiments were carried out to determine the suitability of acidified and antibiotic-supplemented agars for supporting colony formation by cells ofSaccharomyces cerevisiae andSaccharomyces rouxii. Yeasts had been suspended in sucrose syrups buffered at pH 3.0, 5.0 and 7.0, and stored at 4 and 21°C for periods of time ranging to 10 weeks. TheSaccharomyces species were recovered in equal numbers, regardless of the type of enumeration agar. Sucrose, at concentrations up to 60%, protected cells against inactivation during storage, and survival was greater at 21° than at 4°C.     

Cold shock during liquid production increases storage shelf-life of Cryptococcus nodaensis OH 182.9 after air-drying

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60-70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C.     

Efficacy of agar media for enumerating two Saccharomyces species in sucrose syrups

Experiments were carried out to determine the suitability of acidified and antibiotic-supplemented agars for supporting colony formation by cells ofSaccharomyces cerevisiae andSaccharomyces rouxii. Yeasts had been suspended in sucrose syrups buffered at pH 3.0, 5.0 and 7.0, and stored at 4 and 21°C for periods of time ranging to 10 weeks. TheSaccharomyces species were recovered in equal numbers, regardless of the type of enumeration agar. Sucrose, at concentrations up to 60%, protected cells against inactivation during storage, and survival was greater at 21° than at 4°C.     

Studies on the Peanut Seed Vigor Stored in Different Gases

Peanut seeds (water content 6.21% ) were stored at 38–40 ℃ for artificial ageing storage with different gases. N2 or CO2 delayed peanut seed ageing in comparison with air. The rate of seed germination was not affected by storing for 26 weeks under N2 or CO2, but the vitality of seed lost when stored under air. Determination of peanut seed vigor (root length+ axial length)×percentage of germination may precisely show the change in seed quality during ageing. The results indicate the positive correlation between the respiration and subsequent growth in peanut seeds, and the significantly negative correlation between vigor and electrical conductivity or sugar content in the seed extract.