Effects of carbon concentration and carbon-to-nitrogen ratio on growth, conidiation, spore germination and efficacy of the potential bioherbicide Colletotrichum coccodes

The effect of carbon concentration and carbon-to-nitrogen ratio (C:N) as well as their interaction on Colletotrichum coccodes growth and sporulation in submerged flask culture were evaluated. When C:N ratios were held constant, both mycelial dry biomass and spore yield increased with increasing carbon concentration. The specific spore yields (spore yield g⁻¹ carbon), however, were not significantly different for the same C:N ratio in most cases. The highest spore yields (1.3 × 10⁸ spores per ml) were obtained from media containing 20 g per liter carbon with C:N ratios ranging from 5:1 to 10:1. When the C:N ratio was greater than 15:1, spore yields were significantly decreased with increasing C:N ratios. High carbon concentration (20 g L⁻¹) combined with high C:N ratios (above 15:1) reduced both mycelial growth and sporulation, and increased spore matrix production. Spores produced in medium containing 10 g L⁻¹ carbon with C:N ratios from 10:1 to 15:1 had 90% germination on potato dextrose agar after 12 h and caused extensive shoot dry weight reduction on the target weed, velvetleaf. These results suggest that C:N ratios from 10:1 to 15:1 are optimal for C. coccodes spore production. Received 9 December 1997/ Accepted in revised form 22 May 1998     

Effect of ozone on spore germination,spore production and biomass production in two <Emphasis Type="Italic">Aspergillus</Emphasis> species

The ability of ozone gas to reduce food spoilage is relatively well documented, but the developmental effects of the gas on food spoilage fungi are not well known. In this study two model aspergilli, Aspergillus nidulans and Aspergillus ochraceus were used to study the effects of ozone on spore germination, sporulation and biomass production. These effects were examined under three levels of ozone; two high level ozone exposures (200 and 300 μmol mol⁻¹) and one low level exposure (0.2 μmol mol⁻¹). The two species behaved noticeably different to each other. Ozone was more effective in reducing growth from spore inocula than mycelia. No spore production could be detected in A. nidulans exposed to continuous low level O₃, whereas the same treatment reduced spores produced in A. ochraceus by 94%. Overall the work suggests that ozone exposure is an effective method to prevent spread of fungal spores in a food storage situation.     

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.     

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.     

High-density spore production of a <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">cereus</Emphasis> aquaculture biological agent by nutrient supplementation

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 × 10¹⁰ CFU ml⁻¹.     

Verticillium lecanii Spore Formulation Using UV Protectant and Wetting Agent and the Biocontrol of Cotton Aphids

Verticillium lecanii spores (10⁸ spores ml⁻¹) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.     

Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

Influence of culture conditions on production and freeze-drying tolerance of Paecilomyces fumosoroseus blastospores

With the goal of developing a defined medium for the production of desiccation-tolerant blastospores of the bioinsecticidal fungus Paecilomyces fumosoroseus, we evaluated the impact of various media components such as amino acids, carbohydrates, trace metals and vitamins on hyphal growth and sporulation of P. fumosoroseus cultures and on the freeze-drying tolerance of blastospores produced under these conditions. A comparison of 13 amino acids as sole nitrogen sources showed that glutamate, aspartate, glycine and arginine supported biomass accumulations (12–16 mg ml⁻¹) and blastospore yields (6–11 × 10⁸ blastospores ml⁻¹) comparable to our standard production medium which contains casamino acids as the nitrogen source. Using glutamate as the sole nitrogen source, tests with various carbohydrates showed that P. fumosoroseus grew best on glucose (18.8 mg biomass ml⁻¹) but produced similar blastospore concentrations (7.3–11.0 × 10⁸) when grown with glucose, glycerol, fructose or sucrose. P. fumosoroseus cultures grown in media with sodium citrate or galactose as the sole carbohydrate produced lower blastospore concentrations but more-desiccation-tolerant spores. Zinc was the only trace metal tested that was required for optimal growth and sporulation. In a defined medium with glutamate as the nitrogen source, vitamins were unnecessary for P. fumosoroseus growth or sporulation. When blastospores were freeze-dried in the absence of a suspension medium, residual glucose (>2.5% w/v) was required for enhanced spore survival. Thus, a defined medium containing basal salts, glucose, glutamate and zinc can be used to produce optimal concentrations of desiccation-tolerant blastospores of P. fumosoroseus. Received 27 October 1998/ Accepted in revised form 06 May 1999     

The sterol biosynthesis inhibitor molecule fenhexamid impacts the vegetative compatibility of Glomus clarum

The vegetative compatibility of the arbuscular mycorrhizal fungus (AMF) Glomus clarum MUCL 46238 was evaluated after continuous exposure to fenhexamid, a sterol biosynthesis inhibitor (SBI). Three lineages of this AMF were cultured in vitro for five generations in association with Ri T-DNA transformed carrot roots in the presence of 0, 5 or 10 mg l⁻¹ of fenhexamid. Whatever the AMF generation, fenhexamid at 5 and 10 mg l⁻¹ had no significant impact on the number of spores produced. However, vegetative compatibility tests (VCT) conducted with spores from the three lineages, in the presence of 10 mg l⁻¹ of fenhexamid, impacted the anastomosis process. At this concentration, the morphology of the germ tubes was modified. In addition, nitrotetrazolium–trypan blue staining revealed that 10 mg l^−l of fenhexamid significantly reduced the probability of fusion between the germ tubes regardless of the culture conditions (i.e. absence or presence of fenhexamid) preceding the VCT. Our results demonstrated that spore production was not affected by fenhexamid, while anastomosis between germ tubes was decreased. This suggested that high concentrations, accumulation or repeated application of this SBI fungicide may impact the community structure of AMF in soil.     

The morphological and physiological evolution of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> mycelium in the submerged culture and its relation to the formation of secondary metabolites

The influence of the morphology and differentiation of Aspergillus terreus hyphae on the formation of mevinolinic acid (lovastatin) and (+)-geodin was tested. Lovastatin titre was the highest (above 60 mg l⁻¹) in the system with smaller pellets (diameter below 1.5 mm) and high biomass concentration (above 10 g l⁻¹ in the idiophase). These biomass features were induced by the higher initial number of spores in the preculture (above 2 × 10¹⁰ l⁻¹). At the initial number of spores below 2 × 10⁹ l⁻¹ (+)-geodin biosynthesis was the most efficient but it was rather connected with the elevated C/N ratio than with the pellet size. In order to quantify the hyphal differentiation in fungal pellets a special approach was used. The sectioning of the stained pellets together with the image analysis and calculation procedures were applied. The analysis of hyphal differentiation indicated that lovastatin formation was correlated with the fraction of the active, growing hyphae.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation of algal spore lytic C17 fatty acid from the crustose coralline seaweed Lithophyllum yessoense

The algal spore lytic fatty acid of heptadeca-5,8,11-trien (HpDTE: C17:3) was isolated from the crustose coralline seaweed Lithophyllum yessoense. HpDTE, an odd-numbered carbon fatty acid, showed more than 50% lysis at a concentration of 5 μg.mL⁻¹ against the spores of three chlorophyte species, nine rhodophytes, four phaeophytes, and the cells of four phytoplankton species. Lysis activity increased with the number of double bonds and carbon atoms in the fatty acid increased. HpDTE showed a ten-fold stronger activity with a LC₅₀ of 3.1 μg.mL⁻¹ than α-linolenic acid (C18:3).     

Regulation of phosphoglycerate mutase in developing forespores and dormant spores ofBacillus megaterium by thein vivo levels of phosphoglycerate mutase inhibitor

Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn²⁺ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn²⁺ However, the levels of the inhibitor decreased considerably (75–85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn²⁺ and Ca²⁺ as determined by Scatchard plot analysis.     

Dormancy and germination in axillary turions ofHydrilla verticillata

Effects of environmental conditions and growth regulators on release from dormoncy of axillary turions inHydrilla verticillata were investigated. Coll treatment at 2 C for 33 days produced the most complete release from dormancy. One week of 2 C treatment was sufficient for the germination; however, longer cold periods produced more rapid growth in shoot or root lengths as well as a shorter lag time for germination. Dormancy in turions could be broken by a photoperiod of 16 hr but not by on of 8 or 12 hr, nor by continuous lighting. When a cold treatment was applied turions grew out in response to all of the photoperiodic conditions. Red and far-red irradiation during the incubation after a cold treatment promoted gremination; blue and green light markedly inhibited the germination. At 10⁻⁴ and 10⁻⁵ M, gibberellic acid broke dormancy of non-cold treated turions, but was toxic at 10⁻⁴ M to the development after germination. Gibberellic acid promoted growth of cold treated turions even at 10⁻⁶ M. Indoleacetic acid at 10⁻⁴, 10⁻⁵ and 10⁻⁶ M induced outgrowth of both non-cold treated and cold treated turions. Apparently normal growth and development was observed in a high concentration of indoleacetic acid.     

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     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Effects of daylength,irradiance and settlement density on the growth and reproduction of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> gametophytes

The effects of daylength, irradiance and spore settlement density on the growth, maturation and sporophyte production of Undaria pinnatifida (Harvey) Suringar gametophytes were examined using a factorial experimental design in culture. The growth of Undaria gametophytes increased with increasing daylength (8, 12 and 16 h), but the maximum fertility occurred at a daylength of 12 h followed by 8 and 16 h. Gametophytes grew better at the 16 h daylength under the same mean daily irradiance (MDI) of 20 μmol photons m⁻² s⁻¹. However, the fertility was higher at the short daylength (8 h), indicating that the maturation of U. pinnatifida gametophytes is influenced by daylength rather than by the MDI. Vegetative growth and sporophyte production of gametophytes were better at 60 μmol photons m⁻² s⁻¹ than at 30 μmol photons m⁻² s⁻¹ under a 8:16 h LD (Light: Dark) cycle, and their growth and maturation were density-dependant in 16 and 12 h daylength, respectively. These results suggest that the U. pinnatifida gametophytes require a certain amount of light for the growth and reproduction, and intraspecific competition occurred under the optimal growth and maturation conditions. However, the sporophyte recruits per unit has been enhanced with increasing spore settlement density at 8 and 12 h daylengths indicating that high settlement density gives a benefit for maintaining population, even though the sporophyte production of each female plant is inhibited. In conclusion, the vegetative growth, reproduction and sporophyte production of U. pinnatifida gametophytes are retarded at a low irradiance above growth saturation and a high settlement density, and are determined by daylength.     

Light-Dependency of Growth and Secondary Metabolite Production in the Captive Zooxanthellate Soft Coral Sinularia flexibilis

The branching zooxanthellate soft coral Sinularia flexibillis releases antimicrobial and toxic compounds with potential pharmaceutical importance. As photosynthesis by the symbiotic algae is vital to the host, the light-dependency of the coral, including its specific growth rate (μ day⁻¹) and the physiological response to a range of light intensities (10–1,000 μmol quanta m⁻² s⁻¹) was studied for 12 weeks. Although a range of irradiances from 100 to 400 μmol quanta m⁻² s⁻¹ was favorable for S. flexibilis, based on chlorophyll content, a light intensity around 100 μmol quanta m⁻² s⁻¹ was found to be optimal. The contents of both zooxanthellae and chlorophyll a were highest at 100 μmol quanta m⁻² s⁻¹. The specific budding rate showed almost the same pattern as the specific growth rate. The concentration of the terpene flexibilide, produced by this species, increased at high light intensities (200–600 μmol quanta m⁻² s⁻¹).     

Bioreactor for continuous synthesis of compactin by Penicillium cyclopium

Compactin was synthesized by Penicillium cyclopium in submerged as well as in bioreactor systems and assayed spectrophotometrically with a detection limit of 0.5 μg ml⁻¹ solvent. Synthesis in submerged culture was affected by aeration, glucose level, pH, and type and molarity of buffer. Citrate or succinate (pH 4.0, 0.10 M) in malt glucose peptone broth (MGPB) stimulated cell specialization, sporulation, enhanced compactin permeation from mycelia and its production (60.05 μg ml⁻¹ after 12 days). Fungal spores, immobilized onto-into loofah sponge, in a bioreactor, using MGPB-citrate as feed stock, resulted in productivity of 23.04 mg compactin (L⁻¹ h⁻¹) during 50 days operation at 0.45 h⁻¹ dilution rate. Compactin synthesis in the bioreactor was also affected by culture age, substrate, incubation and dilution rates. Scanning electron micrographs of the loofah sponge, prior to, during and post-spores immobilization showed that loofah channels served well for fungal support in the bioreactor. Received 6 October 1997/ Accepted in revised form 2 September 1998     

Control of sporulation in the filamentous cyanobacteriumAnabaena torulosa

In the cyanobacteriumAnabaena torulosa, sporulation occurred even during the logarithmic growth phase. Sporulation was initiated by differentiation of the vegetative cell on one side, adjoining the heterocyst followed by differentiation of the vegetative cell on the other side. Subsequently, spores were differentiated alternately on either side to form spore strings. The sequence of sporulation supports the previous notion that a gradient of spore maturation exists in cyanobacteria and also indicates that the gradient is manifested unequally on either side of heterocysts. Sporulation was absent or negligible in a minerally enriched medium but ocurred readily in a minimal medium. The extent of sporulation was inversely related to phosphate concentration. Sporulation was enhanced at higher temperature. Incandescent light, but not fluorescent light, greatly stimulated sporulation suggesting possible involvement of red light in spore differentiation. Addition of filtrate, from 5 to 8 day old cultures, to freshly inoculatedA. torulosa greatly enhanced sporulation indicating the influence of extracellular products in spore formation.