Preservation, mass production and storage ofStagonospora convolvuli, a bioherbicidecandidate for field bindweed (Convolvulusarvensis)

Different solid substrates were investigated as spore production methods for Stagonospora convolvulistrain LA39, a potential bioherbicide for field bindweed (Convolvulusarvensis L.). Up to 4 × 10⁸ spores/g of substratewere yielded on cous-cous (cracked hard wheat). Thespores were as pathogenic as those grown on artificial medium (V-8-juice agar). The air-drying on kaolin and storage at 3 °C kept spores viable and pathogenic for 180 days. Spore germination exceeded70% for the first 140 days and then declined to 50%after 175 days. Less than 5% of spores were still viable after 17 months. The preservation of stock cultures in 10% glycerine at −80 ° C and in liquid nitrogen did not affect viability orpathogenicity of the spores. This revised version was published online in July 2006 with corrections to the Cover Date.     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

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     

The microbiology of moist barley storage in unsealed silos

The microflora of moist barley grain and whole-crop barley silage stored in top-unloading, unsealed concrete-staved silos on six farms in England depended on the initial water content of the grain (23–58%), method of covering the grain, and the rate at which it was unloaded. Fungi and actinomycetes were fewest when the initial water content was more than 30%, and the grain was covered first with a layer of wilted grass, and then a plastic sheet. During unloading, the uppermost layer of grain remained in good condition provided 7.5 cm was removed daily. With an inefficient top-seal, the top grain heated and became mouldy, as it also did when unloading was slow. As the rate of unloading slowed, heating increased, and a characteristic succession of fungi and actinomycetes developed. With unloading at 7.5 cm/day or more, only yeasts, chiefly Endomycopsis chodatii Wickerham and Hansenula anomala (Hansen) H. & P. Sydow, were abundant, but at slightly slower rates of unloading Penicillium spp. also became common. Both these groups became less common as unloading was slowed further and were replaced, first by Absidia spp. and Mucor pusillus Lindt, then Aspergillus fumigatus Fres., Humicola lanuginosa (Griffon & Maublanc) Bunce, Micropolyspora faeni Cross, Maciver & Lacey, and Thermoactinomyces vulgaris Tsiklinsky as the heating increased. The number of spores (including bacterial cells) that could be removed from samples by blowing air ranged from 0.4–428 times 10⁶/g dry weight of grain. Whole-crop barley silage contained 2.9–132 times 10⁶spores/g dry weight. Similar species were isolated from whole-crop silage as from grain. Little moulding occurred deeper than 30 cm below surface of the grain. Concentrations of airborne spores were estimated periodically during two seasons. There were always more airborne spores than is usual in outdoor air. Without disturbance the silos contained 10⁶-10⁷ spores/m³ air, but when mouldy grain was unloaded concentrations increased to a maximum of 2860times10⁶ spores/m³ air; more than half of these were bacteria and actinomycetes and a quarter Aspergillus flavus Link. Potentially pathogenic fungi and actinomycetes were frequent, particularly when they also occurred in the grain or capping materials for spontaneous heating. Some probably survived in dust deposits and were resuspended during unloading. Airborne spores were frequent around the silos when grain was unloaded and rolled. Workers should wear efficient dust respirators at these times and while inside silos.     

Spontaneous mutants of Microsporon gypseum resistant to griseofulvin

Summary 1) The spores of the microconidial mutant I–18 of the dermatophyteMicrosporon gypseum in agar medium with GF germinated and formed germ tubes deformated in a characteristic way. From 1µg GF/ml up with an increasing antibiotic concentration (expressed in logarithms) the munber of colonies grown (expressed in probits) decreased linearly.2) As a sensitivity measure of the spores the median efficient dose ED 50 was used which was determined by means of a graphic probit analysis. For the strain used this value was determined in the range between 1.35–1.95µg GF/ml in three independent experiments.3) From the smears of a thickened spore suspension (1.6–14.2 × 10⁷ viable spores) in medium containing a high GF concentration a very small, but as for the order a stable number of colonies grew, as found in eight independent experiments. On the medium containing 20µg GF/ml in average 61 colonies grew, on 40µg GF/ml 20 colonies, on 80µg GF/ml 3 colonies and on 160µg GF/ml 0.3 colony (expressed in 10⁷ viable spores tested).4) A part of these colonies were isolated and transferred 29 times on a medium without the antibiotic. Two isolates only show a permanently increased resistance to GF, viz. the strain D-29 which is 50 × more resistant and the strain N-53 which is 3.5 × more resistant than the wild strain I-18.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Trichostomopsis aaronis (Lor.) Agnew & Townsend new to the U.S.S.R.

Abstract

Occurrence of Ptilidium pulcherrimum in transects and spore dispersal from a single colony have been studied in a coastal spruce forest in northern Sweden. The main substrate type was rotting wood with 75% of all occurrences. Annual spore production was 68,500 spores/m² forest, 640,000 spores/m² substrate and 44,000,000 spores/m² colony. Almost 50% of the spores were deposited within 2.5 m of the colony. Annual spore deposition between colonies was estimated to be between 24,000–39,000 and deposition on the main substrate, decaying logs, was about 340–600 spores/m² forest. P. pulcherrimum showed a clumped distribution pattern up to about a 15 m neighbourhood distance. This pattern could not be explained by a similar clumping of the substrate. Instead a limitation by distance in establishment due to a deficit of spores is assumed.     

Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae

The spore productivity and insecticidal activity of two opportunistic insect pathogenic Aspergillus species (namely: Aspergillus clavatus Desmazieres and Aspergillus flavus Link (Ascomycota: Eurotiales, Trichocomaceae)) were compared to Metarhizium anisopliae sensu lato (Metchnikoff) Sorokin (Ascomycota: Hypocreales, Clavicipitaceae) for mosquito (Diptera: Culicidae) control. The production of aerial spores on wheat bran and white rice was investigated in solid-, semi-solid-, and liquid-state media supplemented with a nutritive solution. Wheat bran-based media increased the spore yield in solid-state from three to sevenfold: A. clavatus produced 48.4?±?5.2 and 15.7?±?1.6?×?10⁸ spores/g, A. flavus produced 22.3?±?4.1 and 3.1?±?2.5?×?10⁸ spores/g, and M. anisopliae produced 39.6?±?6.5 and 13.1?±?2.6?×?10⁸ spores/g of wheat bran or white rice, respectively. A. clavatus, A. flavus and M. anisopliae spores harvested from wheat bran-based solid-state media showed lethal concentrations (LC₅₀) of 1.1, 1.8, and 1.3?×?10⁸ spores/ml against Culex quinquefasciatus Say larvae in 72?h. Because A. clavatus and M. anisopliae displayed similar features when cultured under these conditions, our results suggest that insect pathogenic Aspergillus species may be as productive and virulent against mosquito larvae as a well-recognised entomopathogenic fungus.     

Production of Metarhizium anisopliae spores using nutrient‐impregnated membranes and its economic analysis

Metarhizium anisopliae spores were produced on nutrient‐impregnated membranes (NIMs). The NIM system involved wetting the membrane with a spore and nutrient suspension, followed by harvesting the spores produced after incubation. The cost efficiency of spore production was assessed for a range of nutrient sources and membrane types. Skim milk powder (20 g l^‐1) was found to be the most cost‐effective nutrient source of the nine nutrients examined. Yield was 5.7 × 10⁶ spores/cm² after 28 days incubation on a paper membrane. Supplementation of the skim milk with either sucrose (2 g l^‐1) or dextrose + KNO₃ maximized yield. Superwipe, an absorbent fibrous material, was the most efficient of 16 membranes tested which ranged from fibreglass mesh to paper and cloth. A series of small pilot plants were built, but the cost efficiency of spore production decreased as the size of the membrane increased from 24 × 24 cm to 270 × 15 cm and up to 100 × 80 cm. Yield on the two smaller pilot plants was over 10⁷spores/cm², but the cost (nutrient and membrane only) of producing 10¹³ spores (standard dose required per hectare) was around $A37 and was found not to be competitive with spore production on grain.     

Lipid content and fatty acid consumption in zoospores/developing gametophytes of Saccharina latissima (Laminariales, Phaeophyceae) as potential precursors for secondary metabolites as phlorotannins

Phlorotannins are considered inter alia to act protective to a variety of stressors, while lipids in spores are known to fuel various metabolic processes during spore release and settlement. Here, phlorotannin production in zoospores/juvenile gametophytes in relation to lipid metabolism was investigated for the first time in order to study-related metabolic costs. The experiment was carried out in Ny-?lesund (Svalbard, Arctic) within the development from spores to juvenile gametophytes of the brown alga Saccharina latissima over 20 days. In the release stage, the total phlorotannin content of single zoospores was 1.5 × 10⁻⁷ μg and 1.9 × 10⁻⁷ μg in the surrounding medium. Upon release, the total amount of lipids was 1.76 × 10⁻⁵ μg lipid zoospore⁻¹ containing the major fatty acids 16:0 and 18:0, 18:1(n-9), 18:2 (n-6), 18:3(n-3), 20:4(n-6), and 20:5(n-3). During development from spores to gametophytes, a decrease in fatty acids was observed via electron microscopy and a decrease in the fatty acid 18:1(n-9) from 45 to 30% was measured by gas chromatography in particular. While phlorotannin content within the spores remained stable, phlorotannins accumulated in the surrounding medium of gametophytes to 4.0 × 10⁻⁷ μg phlorotannins spore⁻¹ indicating exudation processes. Results obtained support the key and multifunctional role of lipids in zoospore/gametophyte metabolism and may indicate that gametophytes of S. latissima need approximately 10 days to switch to photo-autotrophic strategies, becoming independent of storage lipids. In addition, fatty acids might represent an essential energy source to fuel adaptive responses.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Spore Production Under Solid-State Fermentation of Lignocellulosic Residues

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47?×?10¹⁰ spores g^?1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82?×?10¹⁰ spores g^?1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH₂PO₄, 1 g MgSO₄·7H₂O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105?×?10¹⁰ spores g^?1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10?×?10¹¹ spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

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.     

The impact of nutrition on spore yields for various fungal entomopathogens in liquid culture

Spore yields were measured for various fungal entomopathogens grown in six nutritionally different liquid media with low and high carbon concentrations (8 and 36 g l^–1, respectively) at carbon-to-nitrogen (C:N) ratios of 10:1, 30:1 and 50:1. Six fungi were tested: two Beauveria bassiana strains, three Paecilomyces fumosoroseus strains and one Metarhizium anisopliae strain. Spore yields were examined after 2, 4 or 7 days growth. In general, highest spore yields were obtained in media containing 36 g/l and a C:N ratio of 10:1. After 4 days growth, highest spore yields were measured in the three Paecilomyces isolates (6.9–9.7 × 10⁸ spores ml^–1). Spore production by the B. bassiana isolates was variable with one isolate producing high spore yields (12.2 × 10⁸ spores ml^–1) after 7 days growth. The M. anisopliae isolate produced low spore concentrations under all conditions tested. Using a commercial production protocol, a comparison of spore yields for the coffee berry borer P. fumosoroseus and a commercial B. bassiana isolate showed that highest spore concentrations (7.2 × 10⁸ spores ml^–1) were obtained with the P. fumosoroseus isolate 2-days post-inoculation. The ability of the P. fumosoroseus strain isolated from the coffee berry borer to rapidly produce high concentrations of spores prompted further testing to determine the desiccation tolerance of these spores. Desiccation studies showed that ca. 80% of the liquid culture produced P. fumosoroseus spores survived the air-drying process. The virulence of freshly produced, air-dried and freeze-dried coffee berry borer P. fumosoroseus blastospores preparations were tested against silverleaf whiteflies (Bemisia argentifolii). While all preparations infected and killed B. argentifolii, fresh and air-dried preparations were significantly more effective. These results suggest that screening potential fungal biopesticides for amenability to liquid culture spore production can aid in the identification of commercially viable isolates. In this study, P. fumosoroseus was shown to possess the production and stabilization attributes required for commercial development.     

Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material

Synthesis of amylase by Aspergillus niger strain UO-01 under solid-state fermentation with sugarcane bagasse was optimized by using response surface methodology and empirical modelling. The process parameters tested were particle size of sugarcane bagasse, incubation temperature and pH, moisture level of solid support material and the concentrations of inoculum, total sugars, nitrogen and phosphorous. The optimum conditions for high amylase production (457.82 EU/g of dry support) were particle size of bagasse in the range of 6–8 mm, incubation temperature and pH: 30.2°C and 6.0, moisture content of bagasse: 75.3%, inoculum concentration: 1 × 10⁷ spores/g of dry support and concentrations of starch, yeast extract and KH₂PO₄: 70.5, 11.59 and 9.83 mg/g of dry support, respectively. After optimization, enzyme production was assayed at the optimized conditions. The results obtained corroborate the effectiveness and reliability of the empirical models obtained.     

Dispersal of Septoria nodorum Pycnidiospores by Simulated Raindrops in Still Air

Simulated raindrops, diameter c. 3 or 4 mm, fell 13 m down a raintower onto suspensions of Septoria nodorum pycnidiospores, depth 0.5 mm, or infected straw pieces. Splash droplets were collected on pieces of fixed photographic film. It was estimated that one drop generated c. 300 spore carrying splash droplets, containing c. 6000 spores, from a concentrated spore suspension (6.5 × 10⁵ spores/ml) and c. 25 spore-carrying droplets, containing c. 30 spores, from infected straw pieces (11 × 10⁶ spores/g dry wt). When the target was a spore suspension in water without surfactant, most spore-carrying droplets were in the 200—400 μm size category and most spores were carried in droplets with diameter >1000 μm. When surfactant was added to spore suspensions, most spore-carrying droplets were in the 0–200 μm category and most spores were carried in droplets with diameter 200–400 μm and none in droplets >1000 μm. Regression analyses showed a significant (p < 0.001) relationship between square root (number of spores per droplet) and droplet diameter; the slope of the regression line was greatest when surfactant was added to the spore suspensions. The distribution of splash droplets with distance travelled from the target was better fitted by an exponential model than by power law or Gaussian models. The distributions of spore-carrying droplets and spores with distance were fitted better by an exponential model than by a power law model. Thus regressions of log, (number collected) against distance were all significant (p < 0.01); the slopes of the regression lines were steepest when surfactant was added to the spore suspension. At a distance of 10 cm from target spore suspensions most splash droplets and spore-carrying droplets were collected at height 10–20 cm, with none above 40 cm; at a distance of 20 cm there were most at heights 0–10 cm and 40–50 cm.     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.